Abstract
Chronic obstructive pulmonary disease (COPD) is a major cause of mortality worldwide and is characterized by an excessive airway neutrophilic response. The neutrophil chemoattractant proline-glycine-proline (PGP) and its more potent acetylated form (acPGP) have been found to be elevated in patients with COPD and act via CXCR2. Here, we investigated the impact of neutralizing PGP peptides in a murine model for emphysema. The PGP-neutralizing peptide l-arginine-threonine-arginine (RTR) was used first in a 6-week model of cigarette smoke exposure, where it attenuated lung inflammation. Then, in a model of chronic smoke exposure, mice were exposed to cigarette smoke and RTR treatment was initiated after 10 weeks of smoke exposure. This treatment was continued together with smoke exposure for another 13 weeks, for a total of 23 weeks of smoke exposure. RTR significantly inhibited neutrophil and macrophage influx into the lungs in the 6-week model of exposure. RTR also attenuated the development of emphysema, normalized lung volumes, and reduced right ventricular hypertrophy in the chronic exposure model. Murine epithelia expressed CXCR2, and this expression was increased after smoke exposure. In vitro, human bronchial epithelial cells also demonstrated robust expression of CXCR2, and stimulation of primary human bronchial epithelial cells with acPGP led to increased release of MMP-9 and IL-8. Overall, these results provide evidence that acPGP plays a critical role during the development of emphysema in cigarette smoke–induced injury, and highlight a new epithelial mechanism by which acPGP augments neutrophilic inflammation.
Keywords: COPD, neutrophils, emphysema, ECM fragmentation
Clinical Relevance
This study provides direct evidence for the impact of matrikines (proline-glycine-proline peptides) on the development of smoke-induced emphysema. Furthermore, it highlights the importance of the epithelial CXCR2 receptor as an underappreciated target for airway inflammation. Finally, this study provides a potential therapeutic approach to attenuate proline-glycine-proline peptides in vivo.
Chronic obstructive pulmonary disease (COPD) is a disease state characterized by airflow limitation that is not fully reversible. The airflow limitation is progressive and is also associated with an abnormal inflammatory response of the lungs to noxious particles or gases (1). According to the World Health Organization, COPD has become one of the major causes of mortality worldwide and is predicted to be the third most common cause of death in 2030 (2). The main risk factor for COPD is cigarette smoking, which initiates a neutrophilic inflammatory response (3).
In the last decade, several studies have characterized the collagen-derived neutrophil chemoattractant proline-glycine-proline (PGP) and prolyl endopeptidase (PE) as possible novel biomarkers in COPD. PGP is initially cleaved from collagen by matrix metalloproteases (MMP-8 and MMP-9) and subsequently is cleaved by PE (4–6). Acetylation of PGP (acPGP) further increases its chemotactic activity (7) and protects the peptide from enzymatic degradation by the aminopeptidase activity of leukotriene A4 hydrolase (LTA4H) (8), thereby leading to a more stable inflammatory molecule. Unfortunately, to date, the mechanism by which PGP becomes acetylated remains unknown.
Various studies have linked increased production of acPGP with emphysema. For instance, mice exposed to chronic cigarette smoke exposure were found to develop an emphysema-like phenotype and have elevated pulmonary PGP levels (8, 9). Paige and colleagues showed that smoke-exposed mice treated with the pharmaceutical agent 4-methoxydiphenylmethane, which selectively augments LTA4H aminopeptidase activity without affecting the bioproduction of leukotriene B4, over a 6-month period prevented the development of emphysema (10). Furthermore, in clinical studies, significantly higher acPGP levels were detected in airway and blood samples taken from individuals with COPD compared with smokers without COPD and healthy volunteers (4, 9, 11).
Although the proximal mechanisms leading to this increased proteolytic degradation of collagen are unknown, stimulation of airway epithelial cells by cigarette smoke or noxious particles is believed to be a significant contributor (8). In a previous study, we showed that when the generation of PGP by valproic acid was inhibited in an acute 5-day smoking model, cigarette smoke–induced neutrophil influx was significantly reduced (12). We hypothesized that PGP peptides are critical in the development of smoke-derived emphysema. Here, we used the PGP-neutralizing peptide l-arginine-threonine-arginine (RTR) to inhibit both inflammation and heart remodeling in a 6-week model of smoke exposure (13). Results from a 23-week model of smoke exposure showed that RTR has potential utility as a therapeutic. Finally, in vitro experiments suggested that PGP can signal the release of both MMP-9 and IL-8 from airway epithelia, highlighting a new mechanism whereby PGP could augment neutrophilic inflammation.
Methods
In Vivo Studies
Murine smoking models
All of the animal experiments conducted in this study were reviewed and approved by the Institutional Laboratory Animal Care and Use Committee of the University of Alabama at Birmingham (UAB; animal protocol #120709133) and conformed to the National Institutes of Health “Guidelines for the Care and Use of Laboratory Animals” (14). Two murine smoke models were used. Due to the risk of aggressive behavior by male mice in long-term experiments, female mice were used in both models. In a subchronic model, female BALB/c mice (8–10 wk old; The Jackson Laboratory) were randomly assigned to either smoke or air exposure over 5 days a week for 6 consecutive weeks (n = 4–6 mice per group). Mice were exposed for 60 minutes per day to mainstream cigarette smoke from standard University of Kentucky 3R4F research cigarettes (9.4 mg tar/0.726 mg nicotine). Cigarette smoke was generated and delivered by the InExpose smoking system (SCIREQ) according to a previously established protocol (15). Before each exposure, the mice received either vehicle (50 μl saline), RTR (50 μg in 50 μl saline), or the control peptide alanine-serine-alanine (ASA) (50 μg in 50 μl saline). Therapeutic agents were administered by oropharyngeal aspiration under light isoflurane anesthesia to directly reach the lungs.
In a murine model of chronic smoke exposure, mice of the same strain and age as described above were exposed to smoke and treated during 23 weeks. In this experiment, 70 mice were randomly divided into three groups: a cigarette smoke–exposed group treated with vehicle (n = 40), and air-exposed groups that received either vehicle (n = 15) or RTR (n = 15). After 10 weeks of smoke exposure, the 40 smoke-exposed and vehicle-treated mice were then divided into two groups: a smoke-exposed group that still received vehicle (n = 20) and a smoke-exposed group that underwent RTR rescue (n = 20). Before the mice were killed, the lung volumes of seven to eight mice per group were measured with a flexiVent (SCIREQ). Sixteen hours after the last smoke exposure, the mice were killed to collect lung tissue for morphometry (16, 17), BAL fluid (BALF), and blood. Specific murine-related analyses are addressed in the data supplement.
Ex Vivo Studies
Airway epithelial cells
All epithelial cell lines (wild-type CFBE [cystic fibrosis bronchial epithelial], F508 CFBE, 16HBE14o-, Calu3, and HeLa) were provided by the UAB Cystic Fibrosis Research Center. Primary human bronchial epithelial (HBE) cells were derived from lung explants after written informed consent was obtained from individuals with and without COPD. During 4 weeks, the cells were grown and terminally differentiated in polarizing conditions in inserts. The inserts with primary airway epithelia underwent immunofluorescence staining or in vitro stimulation as described in the data supplement. The use of human cells and tissues was approved by the UAB Institutional Review Board.
Statistics
When appropriate, as the data were normally distributed, an unpaired t test was used, or one-way ANOVA followed by the Newman-Keuls multiple-comparison test was performed using GraphPad Prism 6. P ≤ 0.05 was considered statistically significant.
Results
To establish a potential link between the early development of emphysema and inflammation with PGP peptides, mice were exposed to cigarette smoke for 6 weeks. At the end of the experiments, BALF was collected. As expected, exposure to smoke led to increased leukocyte influx into the lungs.
Treatment with RTR decreased the total cell influx measured in the BALF by 63% compared with treatment with the control peptide (ASA) and by 66% compared with vehicle (Figure 1A). The cell influx after smoke exposure was due to an increased number of macrophages and neutrophils, which was significantly attenuated after RTR treatment, but not after treatment with the control peptide (Figures 1B and 1C). These findings are associated with acPGP peptide levels measured in BALF (Figure 1D).
Figure 1.
RTR attenuates inflammation in a 6-week murine smoke-exposure model. RTR was administered to mice intratracheally 5 days a week over 6 weeks before they were exposed to smoke. The tripeptide ASA was used as a control. (A) RTR significantly inhibited leukocyte influx into the lungs. (B and C) More specifically, macrophage (B) and neutrophil (C) influx was significantly decreased. (D) The tripeptide acPGP was significantly increased in the BALF of smoke-exposed mice and returned to baseline levels after RTR treatment. In all cases, the ASA-treated/smoke-exposed mice did not significantly differ from the smoke-exposed control group. Data are shown as the mean ± SEM (n = 5–6 mice per group). Statistical analysis was performed by one-way ANOVA followed by the Newman-Keuls multiple-comparison test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; #not significant (saline-treated/smoke-exposed vs. RTR-treated/smoke-exposed). acPGP = acetylated proline-glycine-proline; ASA = alanine-serine-alanine; BALF = BAL fluid; ns = not significant; RTR = l-arginine-threonine-arginine.
We next sought to study the potential for targeting PGP peptides in an murine model of emphysema. Mice were exposed to cigarette smoke for 23 weeks, after which the BALF and airways were collected for morphometry. Mice exposed to smoke had elevated total cell influx in the BALF (Figure 2A). As was observed in the 6-week model, this increased number was due to infiltration of macrophages and neutrophils (Figures 2B and 2C). RTR treatment caused a significant attenuation of the total cell counts of both neutrophils and macrophages, comparable to those observed in the air-treated mice. As was observed in the short-term smoke-exposure group, the acPGP levels were increased by smoke exposure and were significantly reduced by RTR treatment in the BALF (Figure 2D).
Figure 2.
RTR attenuates inflammation in a 23-week murine smoke-exposure model. Mice were initially exposed to cigarette smoke for 10 weeks (5 d/wk) without RTR treatment, and then RTR was administered intratracheally over the final 13 weeks of smoke exposure in the mice to develop a therapeutic approach (RTR rescue). (A) RTR treatment caused a significant drop in total cell influx into the lungs. (B and C) This was caused by an inhibition of the influx of macrophages (B) and neutrophils (C). (D) acPGP levels were decreased after RTR therapy. Data are shown as the mean ± SEM (n = 8–12 mice per group). Statistical analysis was by one-way ANOVA followed by the Newman-Keuls multiple-comparison test. *P < 0.05, **P < 0.01, and ****P < 0.0001.
After the lungs were sectioned for morphometry, increased alveolar enlargement after smoke exposure was observed (quantified in Figure 3A; representative images are shown in Figure 3E). RTR treatment attenuated alveolar enlargement caused by chronic smoke exposure (a consequence of emphysema development) in a therapeutic strategy. Preservation of the lung architecture was accompanied by maintenance of pulmonary function, as measured by the inspiratory capacity (Figure 3B), and a trend toward a reduction in compliance (Figure 3C). These effects were associated with the development of right ventricular hypertrophy (RVH), which was significantly improved by RTR rescue (as shown in Figure 3D) but did not resolve to baseline. Overall, these results strongly indicate a central role for PGP peptides in experimental emphysema.
Figure 3.
RTR attenuates histological and physiological changes in a 23-week murine smoke-exposure model. (A and E) Representative lung slices (E) demonstrate that RTR attenuated the mean linear intercept (A). (B–D) RTR treatment also normalized the inspiratory capacity (B), reduced static compliance (C), and attenuated right ventricular hypertrophy (D). Data are shown as the mean ± SEM (n = 8–12 mice per group). Statistical analysis was performed by one-way ANOVA followed by the Newman-Keuls multiple-comparison test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Scale bars: 50 μm. Lm = mean linear intercept (chord) length; LV = left ventricular; RV = right ventricular; S = septum.
When the murine tissues were examined for CXCR2 expression, increased CXCR2 expression was observed in the airway epithelia of all of the mice (for a representative example, see Figure 4A). When smoke-exposed mice were compared with air-exposed mice, an approximately 60% increase in epithelial CXCR2 expression was observed (Figure 4B). These findings led to the identification of CXCR2 expression in various human airway epithelial cell lines and via RT-PCR (Figure 4C), and CXCR2 expression on wild-type CFBE by immunohistochemistry (Figure 4D). Primary HBE cells expressed CXCR2 with an apical localization (Figure 4E).
Figure 4.
CXCR2 expression is observed in murine epithelia, multiple airway epithelial cell lines, and primary airway epithelial cells. (A) Murine lungs express CXCR2 on airway epithelia relative to the IgG control (panel and inset). (B) Representative histology (upper panels) of non–smoke-exposed versus smoke-exposed murine lung CXCR2 staining; the quantitative intensity of CXCR2 expression is shown in the lower panel. (C and D) Cxcr2 gene expression is observed in multiple airway epithelial cell lines (C), as well as in primary human airway epithelial cells and HeLa cells (D). (E) Upper panel: immunofluorescence of WT CFBE cells demonstrating CXCR2 expression (green) relative to the nucleus (blue) and cell membrane (red); lower panel: representative immunofluorescence of primary airway epithelial cell demonstrating CXCR2 expression (green) relative to the nucleus (blue), with a Z-stack demonstrating apical CXCR2 expression. Scale bar: 100 μm. a.u. = arbitrary unit; Calu-3 = human lung cancer cell line; CFBE = cystic fibrosis bronchial epithelial; Ctrl = control; HBE = human bronchial epithelial; WT = wild-type.
After exposing primary HBE cells from patients with COPD or healthy donors to acPGP, we observed an increased release of IL-8 and MMP-9. Preincubation of acPGP with RTR nullified the effects seen with acPGP (Figures 5A and 5B). It was notable that primary airway epithelial cells from patients with COPD secreted almost two times more IL-8 than those obtained from donors without lung disease. MMP-9 could only be detected in the supernatant of the acPGP-stimulated primary airway epithelia from patients with COPD.
Figure 5.
Airway epithelial cells respond to CXCR2 stimulation. (A and B) IL-8 (A) and total MMP-9 (B) levels were measured via ELISA in the supernatant of primary HBE cells from patients with COPD and donors without lung disease. Cells were incubated for 8 hours with acPGP (500 μg/ml), RTR (50 μg/ml), or a combination of both. Statistical analysis was done by unpaired t test (#P < 0.05) and one-way ANOVA followed by the Newman-Keuls multiple-comparison test (*P < 0.05 compared with the acPGP group, $P < 0.05 compared with the acPGP group, and **P < 0.01 compared with the acPGP group). COPD = chronic obstructive pulmonary disease; MMP-9 = matrix metalloprotease 9.
Discussion
In this report, we describe the role of PGP after subchronic and chronic exposure of mice to cigarette smoke. Neutralizing PGP using RTR resulted in lower levels of pulmonary inflammation and abrogated emphysema development. Furthermore, we provide in vitro evidence that airway epithelial cells can respond to PGP peptide stimulation via CXCR2, highlighting this cell as a potential source to drive matrikine-mediated lung inflammation. This work closely aligns with a recent study by Patel and colleagues in which they described the ability of PGP peptides to act on airway epithelial CXCR2, leading to an enhanced asthma phenotype (18).
In the subchronic smoke-exposure murine model, RTR was superior to either vehicle or the control peptide ASA in reducing the inflammatory response to cigarette smoke exposure. In contrast to RTR, ASA did not reduce the cigarette smoke–related inflammatory response in mice, indicating the specificity of the RTR effect. As can be noted in Figure 1D, the acPGP levels in the BALF followed a trend similar to that observed for the leukocyte numbers. Although it was not statistically significant, there was a trend toward reduced acPGP in saline-treated, smoke-exposed animals compared with the RTR-treated, smoke-exposed group (P = 0.08). Together, these results underscore the potential for the development of RTR as a biologic inhibitor of the polymorphonuclear neutrophil inflammation and ongoing matrix remodeling observed in cigarette smoke–induced injury. In the chronic model, smoke-exposed mice were treated therapeutically with RTR at 10 weeks after start of the cigarette smoke exposure and onset of inflammation. In this model, metrics of inflammation and emphysema were abated by the use of RTR rescue. We hypothesize that the lower macrophage numbers observed in the BALF after RTR treatment are a result of a reduced inflammatory burden due to lower PGP levels.
Lower levels of PGP peptides after RTR treatment in mice were also associated with a loss of PE activity (see the data supplement). This suggests a potential mechanism whereby a reduction in the peptide burden may have an impact on the inflammatory cascade that leads to progressive PE release in vivo.
The attenuation of PE by PGP peptide neutralization indicates that this peptide may play a regulatory role in its ongoing generation.
Because the development of RVH is a known comorbidity in patients with COPD, we investigated how RTR treatment affected RVH in chronically smoke-exposed mice (19). As depicted in Figure 3D, RVH was significantly improved after RTR treatment. It is quite interesting that RTR treatment had a more profound effect on experimental emphysema than on RVH. Although this might be due simply to the intratracheal administration of the RTR, it may suggest that the development of emphysema and and the development of RVH in this model are two parallel processes, with RTR more effectively treating the airway remodeling. Altogether, these results demonstrate that CXCR signaling and bioactive matrix fragments play a critical role as mediators of emphysema, and highlight the potential for targeting these fragments in COPD lung disease.
Our data also highlight the potential of acPGP to induce the release of key mediators, which augments the airway inflammatory response and extracellular degradation. Interestingly, airway epithelial cells from patients with COPD showed an enhanced response to PGP peptides, which could indicate a general propagation of CXCR2 signaling in these cells. Importantly, these results clearly demonstrate that PGP peptides are capable of stimulating epithelial cells and may promote ongoing inflammation in COPD.
To our knowledge, this is the first report to describe that a therapeutic intervention directed toward PGP peptides in a murine smoke-induced emphysema model reduced airway inflammation and development of the morphological and physiological signs of emphysema. These results also demonstrate the effect of acPGP on CXCR2 signaling from airway epithelial cells, leading to augmented inflammation. Our data strongly underscore an important role for PGP in the development of emphysema and epithelial cell dysfunction in COPD.
Supplementary Material
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Footnotes
Supported by a Mosaic grant from the Netherlands Organization for Scientific Research 017.008.029 (M.A.R.); National Heart, Lung, and Blood Institute grants 5R35HL135710-03 (J.E.B.), R01 HL102371 (A.G.), R01 1R01HL126596 (J.E.B. and A.G.), and K08 HL123940 (J.M.W.); VA Merit Review grant 1I01BX001756 (A.G.); and CF Foundation Award BRATCH16I0 (P.E.B.).
Author Contributions: M.A.R., X.X., T.H.A., M.S., T.S., P.E.B., L.V., and C.M.M. conducted experiments. M.A.R., G.M.S., J.M.W., F.A.R., G.F., J.E.B., and A.G. analyzed data. M.A.R., J.E.B., and A.G. wrote the manuscript.
This article has a data supplement, which is accessible from this issue’s table of contents at www.atsjournals.org.
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