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* Division of Pharmacology and Pathophysiology, Department of Pharmaceutical Sciences, Faculty of Science, Utrecht University, Utrecht, The Netherlands; and
Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Alabama, USA
1Correspondence: Division of Pharmacology and Pathophysiology, Department of Pharmaceutical Sciences, Faculty of Science, Utrecht University, P.O. Box 800 82, 3508 TB Utrecht, The Netherlands. E-mail: a.h.vanhouwelingen{at}uu.nl
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: collagen breakdown • COPD • chemotaxis • neutrophil • inflammation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. It is predicted to be the third leading cause of death worldwide in 2020, as stated by the World Health Organization. The prevalence and morbidity is projected to increase even within the next few years. Cigarette smoke is the major (avoidable) risk factor in the development of COPD in developed countries (1)
. On a worldwide scale, smoking is a heavy burden on public health, with a major socioeconomic cost because of the chronicity of COPD (2)
.
Alveolar damage, characterized by loss of airway function, is one of the main features observed in emphysema patients. In this process, both neutrophils and macrophages play an important role. Enhanced levels of macrophages and neutrophils are present in broncho-alveolar lavage (BAL), sputum samples, and airway tissue of COPD patients (3
4
5
6)
. Moreover, mediators released from the neutrophil as well as the macrophage can cause damage to lung tissue, thereby inducing symptoms observed in COPD patients (3
, 5
, 7
, 8)
.
During lung matrix degradation, collagen fibers liberated from the matrix by matrix metalloproteinases (MMPs) are broken down into smaller fragments consisting of proline and glycine repeats. Recently we showed that the collagen-derived peptide N-acetyl-proline-glycine-proline (PGP) is chemotactic for neutrophils via CXCR1 and CXCR2 chemokine receptors and that the PGP or GP sequence is an important motif present in many CXCR ligands in different species (9)
. In vivo, PGP is produced acutely after airway lipopolysaccharide (LPS) exposure, whereas airway PGP administration causes both an acute neutrophil infiltration as well as emphysematous changes when administered chronically. This suggests that PGP can actively recruit neutrophils into the site of inflammation acutely (e.g., lungs) and can maintain inflammatory cells in the tissue when other chemoattractants or chemokines such as interleukin (IL) -8 are absent, thereby driving chronic disease as implied by our detection of PGP in the lungs of COPD patients (9)
.
Because PGP can induce migration of neutrophils, PGP antagonism could be beneficial in the treatment of COPD, especially lung emphysema. Using the complementary peptide design algorithm, the antagonist L-arginine-threonine-arginine (RTR) was created having the sequence Arg-Thr-Arg, which is tetramerized on a dilysine core. RTR was earlier shown to be a potent inhibitor of PGP-induced neutrophil polarization and chemotaxis in vitro (10
, 11)
. Moreover, in vivo RTR inhibited ulceration in the alkali-injured rabbit cornea (10)
.
Based on these observations that RTR inhibits both in vitro and in vivo PGP-induced neutrophil migration, we have investigated the possible therapeutic effects of RTR in a model for lung emphysema. Because PGP is active on neutrophils via CXCR1 and CXCR2 chemokine receptors and shares homology with chemokine agonists of these receptors, we evaluated RTR’s ability to block chemokine-dependent activity on neutrophils as well as PGP’s effects. Therefore, we performed in vivo and in vitro studies with the RTR compound and showed that RTR is able to inhibit in vitro polymorphonucleocyte (PMN) chemotaxis to PGP as well as the cognate CXCR1 and CXCR2 ligand, IL-8. In vivo, RTR treatment inhibits the neutrophilia in mouse airways induced by PGP and prevents neutrophil infiltration in response to the more general inducer of inflammation, LPS. Moreover, RTR attenuated emphysematous changes in mouse airways seen in response to chronic treatment with either PGP or LPS.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Induction of acute inflammation
LPS, PGP (Anaspec, San Jose, CA, USA), or control fluids (PBS) were administered intratracheally in volumes of 50 µl under ketamin-xylazin anesthesia. After 24 h, the animals were sacrificed to perform leukocyte accumulation studies in the airways.
Leukocyte accumulation in BAL fluid
Airways of mice were lavaged 24 h after PGP or LPS treatment. The leukocyte count was determined by hemocytometry. The absolute numbers of neutrophils was established by examination of air-dried cytospins that were stained with hematoxylin and eosin (Diff-Quick; Merz & Dade, Düdingen, Switzerland).
Peroxidase activity in BAL fluid
Myeloperoxidase (MPO) activity was assayed in BAL fluid samples from animals by measuring the change in optical density (OD) using 3,3,5,5-tetramethylbenzidine (TMB) and H2O2. The oxidation of TMB was stopped after 15 min of incubation by addition of H2SO4. The color formation was measured spectrophotometrically by an increase in OD at 450 nm using 550 nm as a reference. The results were expressed as change OD per ml BAL fluid.
Induction of lung emphysema
To induce lung emphysema, PGP (250 µg/mouse), LPS (5 µg/mouse), or control (PBS, 50 µl/mouse) was administered twice a week for a period of 8 wk. PGP, LPS, or control fluids (PBS) were administered intranasally to animals, which were anesthetized with halothane (inhalation of 3% halothane). After 8 wk of treatment, there was a 2-wk recovery period to eliminate the direct effects of PGP and LPS administration.
Morphometric changes in lung tissue
Alveolar septal wall destruction was measured via a standard morphometric technique. Briefly, lungs were dissected en block from the animals and pressure perfused at 25 cm Hg with Carnoy’s solution. After processing and embedding with butyl methacrylate/methyl methacrylate (BMA/MMA), 5 µm sections were cut and stained with hematoxylin and eosin to evaluate morphology. The extent of alveolar damage was measured using digitized images of representative fields not containing blood vessels of bronchi. On the images a grid was superimposed, and the number of intersections with alveolar walls was counted with a custom-made script for the Image Pro 4.0 software package (Media Cybernetics, Silver Spring, MD, USA). The mean linear intercept (Lm) was calculated by dividing the total grid length by the number of intersections. An increase in Lm was taken as an evidence of alveolar enlargement.
Right ventricular hypertrophy measurement
The right ventricle (RV) was dissected free from the left ventricle and septa (LV+S) by using a binocular dissecting microscope. After blotting dry, both ventricles were weighted. The ratio between RV and LV + S is an index for right ventricular hypertrophy.
Chemotaxis assay
Human PMNs were isolated by density centrifugation using histopaque, and chemotaxis experiments were performed. IL-8 and PGP were placed in the bottom wells of a 3 µm 96-well polycarbonate filter plate (Millipore, Billerica, MA, USA) with a 3 µm pore size, with 2 x 105 cells added to the top well. After 1 h of incubation at 37°C in 5% CO, the upper wells were removed, and micrographs of migrated cells were made with an Olympus IX70 microscope (Olympus, Tokyo, Japan) and Perkin Elmer UltraView software (Perkin Elmer, Waltham, MA, USA). The chemotactic index was calculated by dividing cells per high-powered fields in the experimental condition by that seen in the control condition (spontaneous migration).
Antagonist studies
The neutralizing PGP antagonist RTR was synthesized by Anaspec and administered in vivo either intratracheally or intranasally (50 µg/mouse in 50 µl). In the acute inflammation model, RTR was administered on different time points before or after treatment with PBS, PGP, LPS, or alone, depending on the experimental procedure. RTR was administered simultaneously with PBS, PGP, or LPS in the chronic model. In the in vitro experiments, PGP and IL-8 were incubated with RTR for 1 h before PMN chemotaxis.
Data analysis
All data are expressed as mean ± SE and analyzed via a Student’s t test when comparing 2 sets of data. ANOVA was used for comparing of more than 3 groups, and the differences between groups were analyzed by a Bonferroni post hoc test. The cellular accumulation in the BAL fluid was analyzed by using a distribution-free Kruskal-Wallis one-way ANOVA, and the results are expressed as scatter plot with median values or as a bar plot with mean values. All values of P < 0.05 were considered to reflect a statistically significant difference. Analyses were performed by the usage of GraphPad Prism (version 3.0; GraphPad, San Diego, CA, USA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, RTR was able to inhibit both PGP-induced (Fig. 1B
) and IL8-induced (Fig. 1A
) chemotaxis, as measured by the migration assay. The effect of RTR on IL8 is concentration dependent (Fig. 1A
), as it is with PGP (data not shown). Moreover, high concentrations of RTR itself had no influence on baseline cell migration.
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PGP induces a time- and concentration-dependent airway neutrophilia
Based on the in vitro finding that PGP induces migration of neutrophils, we treated BALB/c mice intratracheally with PBS, PGP, or LPS (as a positive control) and found that both PGP and LPS induced migration of inflammatory cells, predominantly neutrophils, into the airway lumen (Fig. 2
A, B). This effect of PGP on the migration of neutrophils was dose dependent (Fig. 2D
) and time dependent (Table 1
). The increase in the number of neutrophils was accompanied by an increase in neutrophil activity, as measured by MPO content in the BAL fluid (Fig. 2C
).
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RTR inhibits acute neutrophilic inflammation induced by PGP
To study the effect of the antagonist RTR on PGP-induced neutrophil migration in mice, we used different treatment protocols with RTR. As can be observed in Fig. 3
A, RTR itself had no influence on basal cell population when the cells were harvested 24 h after RTR application. When RTR was applied simultaneously with PGP, RTR inhibited the PGP-induced migration of neutrophils (Fig. 3B
). Moreover, applying RTR 15 min before (Fig. 3C
), it still inhibited the PGP-induced neutrophilia. Application of RTR 24 h before PGP diminished the inhibitory effect of RTR (Fig. 3D
).
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RTR reduces emphysema-like changes in the airways
Emphysema-like changes in the airways develop after repeated PGP or LPS (Figs. 4
-6
). These emphysema-like changes involve alveolar enlargement as measured by Lm and right ventricular hypertrophy. To interfere in the development of these emphysema-like changes, mice were treated with RTR before exposing them to either PGP or LPS (positive control). RTR inhibited the increase in Lm values induced by either PGP (Fig. 4A
) or LPS (Figs. 5A
and 6)
. Moreover, RTR attenuated both LPS- and PGP-induced right ventricular hypertrophy (Figs. 4B
and 5B
, respectively). As can be observed in Fig. 5
, RTR itself had no influence on baseline lung morphology or hypertrophy.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 12
, 13)
. Moreover, the migrated neutrophils are active (as measured by MPO release) when present at the site of inflammation. This study proves again that PGP has chemotactic activities but also shows that these migrated neutrophils are active at the site of inflammation.
Neutrophilic inflammation is a key factor in emphysema, in which the progression of the disease has been associated with the severity of the inflammation. Enhanced levels of neutrophils were found in both induced sputum and BAL fluids samples of patients with COPD while macrophage counts were unchanged (14)
. Moreover, depletion of neutrophils by antibody treatment reduced an acute inflammatory reaction in mice induced by cigarette smoke, which is the main avoidable risk factor for emphysema (15)
.
Neutrophils play a key role in the lung matrix breakdown. During this process extracellular matrix proteins (e.g., collagen) can be liberated from the network of collagen, which eventually leads to the formation of smaller peptide fragments, specifically the tripeptide PGP. Matrix degradation can be initiated by MMP released by either neutrophils or macrophages on stimulation with cigarette smoke. Recent studies showed that MMP-9 is associated with the production of PGP. MMP-9–/– mice showed decreased PGP BAL fluid levels after Francisella tularensis infection (16)
. However, MMP-9 alone is not enough to induce PGP. Also, propyl endopeptidase released by leukocytes (e.g., macrophages or T cells) is needed for the formation of PGP (17)
.
Based on our previous study, we further hypothesized that PGP is a crucial factor in the development of emphysema (9)
. To test this hypothesis, we have used different models for COPD that have 2 characteristics observed in COPD patients, namely, destruction of airway walls (as measured by Lm) and right ventricular hypertrophy. The first model is based on the finding that LPS is present in large quantities in cigarettes, as well as in cigarette smoke compared to ambient air (17)
. As already demonstrated, multiple dosages of LPS induced emphysema-like changes in the lungs of mice (18)
. Also, our data show that LPS induces alveolar enlargement and right ventricular hypertrophy. We recently showed that PGP is involved in LPS-mediated acute inflammation in the lungs (9)
. This suggests that the LPS model we used for the induction of lung emphysema is also driven in part by PGP.
Because PGP formation is downstream of LPS administration, the second model for emphysema is based on the application of PGP itself. Multiple administrations of PGP lead to alveolar enlargement and right ventricular hypertrophy. The presence of PGP in relation to COPD is not restricted to the animal situation. PGP can be detected in BAL fluids of patients with evidence of COPD, as measured by a positive computed tomography scan and low FEV1 (forced expiratory volume in 1 s) score (9)
. Moreover, PGP can be detected in sputum of patients with cystic fibrosis, which is a chronic neutrophilic inflammation of the lungs (19)
. Collectively, these data suggest that the collagen-derived peptide PGP is involved in neutrophil migration and concomitant development of COPD, especially in lung emphysema.
Because PGP might be involved in the development of emphysema, antagonism of PGP could be beneficial in the treatment of COPD. Therefore, we synthesized an inhibitory compound via the molecular recognition theory. Using this approach, the tetrameric peptide RTR has been designed to bind and neutralize PGP (10
, 11)
. RTR is effective in attenuating the migration of neutrophils both in vitro and in vivo. In vitro, this inhibitory effect is present for PGP as well as CXCL8. In vivo, RTR inhibits the migration of neutrophils induced by PGP. Moreover, RTR inhibits the alveolar damage and heart hypertrophy induced by PGP and LPS. RTR’s antagonism of the development of LPS-dependent emphysema may be important. Direct airway administration of LPS induces global inflammation by immediate activation of resident alveolar macrophages, which release protease, reactive oxygen species, chemokines, and cytokines. The ensuing tissue response leads to the massive infiltration of neutrophils along with the production of matrix breakdown products, such as PGP, and ultimately results in structural breakdown and functional decline of the tissue. The inhibitory effects of RTR on PGP and IL-8 chemotaxis in vitro as well as its ability to suppress PMN infiltration into mouse airways in response to both PGP and LPS suggest that its antagonism of both the CXCR ligands make it capable of preventing the development of the emphysematous phenotype during chronic exposure to PGP and LPS. Our observations suggest that neutralizing PGP and chemokine CXCR ligands seems beneficial in the treatment of emphysema and can be a clue to a possible novel therapeutic approach for COPD.
In conclusion, our studies have provided evidence that PGP may be of importance in the pathogenesis of COPD, especially in patients in which the disease progress is related to a neutrophilic inflammation. Moreover, administration of RTR or other CXC ligand antagonists may prevent or ameliorate the clinical manifestations of COPD. Finally, the development of new compounds with spectra of activity similar to RTR’s may represent a novel class of antiinflammatory agents with utility in a wide range of clinical conditions of multiple organ systems.
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ACKNOWLEDGMENTS |
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Received for publication August 14, 2007. Accepted for publication May 15, 2008.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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