Abstract
Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of death worldwide and has few effective therapies. It is characterized by anomalous and persistent inflammation, both local and systemic. Neutrophilic inflammation predominates in the COPD airway wall and lumen, but, despite the presence of abundant innate immune cells, the progressive clinical course of the disease is punctuated by recurrent infection-driven exacerbations. An extensive body of evidence (from cell culture to murine models and finally to the susceptibility of human patients with α1-antitrypsin deficiency to develop COPD) implicates neutrophil elastase and other neutrophil-derived proteases as key mediators of the tissue damage and relentless decline in lung function that occurs in this condition. In addition to the well recognized role of cytokines in modulating neutrophil function and survival, it has recently become apparent that hypoxia can influence neutrophil function, with impaired killing of pathogenic bacteria, enhanced release of proteases, and delayed apoptosis. This destructive neutrophil phenotype is predicted to be highly detrimental in the setting of the COPD microenvironment.
Chronic obstructive pulmonary disease (COPD) is a major cause of chronic morbidity and is the fourth leading cause of death worldwide (1). The inflammatory response to cigarette smoke is the major etiological factor in the pathogenesis of COPD (2), and even after smoking cessation a continuous cycle of inflammation leads to a continued decline in lung function. Early epidemiological studies suggested that only 15 to 30% of smokers develop COPD (3), although a more recent study estimated that up to 50% of elderly smokers have spirometric evidence of COPD (4). Despite intense efforts to identify genetic predispositions to COPD in response to environmental insults, most identified genetic variants contribute only nominally to overall disease risk. Of those genetic links discerned to date, many have links to inflammation, oxidative stress, the protease/antiprotease balance, and immunosenescence (5, 6).
COPD is characterized by aberrant and persistent inflammation, both local and systemic, leading to progressive decline in lung function and ultimately to hypoxic respiratory failure, cor pulmonale, and death. Acute exacerbations, often triggered by viral or bacterial infection, are superimposed on this chronic illness and lead to further cycles of inflammation, hypoxia, and airway damage. Current therapies have little impact on airway inflammation or disease progression. Recent advances in our understanding of the aberrant inflammatory and tissue repair processes that drive COPD pathogenesis may facilitate the introduction of more effective treatment strategies.
Neutrophils are our first line of defense against microbial invasion and are rapidly recruited to sites of infection. Their primary role is to ingest (phagocytose) invading microorganisms and destroy them by the internal generation of reactive oxygen species and the action of preformed proteases such as neutrophil elastase (NE), cathepsins, and many other antibacterial proteins (7). The neutrophil-derived reagents that combat infection are also strongly implicated in tissue injury in COPD. Neutrophils are the most abundant inflammatory cells present in the bronchial wall and lumen of patients with COPD (8–11), but recurrent infections and persistent bacterial airway colonization drive further inflammatory change. Specific neutrophil degranulation products like NE have long been implicated in tissue damage in this setting, but in recent years there has been a revolution in neutrophil biology, with a new understanding of a more complex repertoire of neutrophil functional responses, including priming, response to hypoxia, cytokine and chemokine release to orchestrate ongoing inflammatory responses, and transdifferentiation into dendritic-like cells with the capacity for antigen presentation. Furthermore, novel pathways of cell death and survival have been explored, including NETosis and autophagy. Innovative imaging modalities have enabled the tracking of neutrophils in living organisms (both in animal models and in human disease), and there is renewed interest from the pharmaceutical industry in the potential for modulating neutrophilic inflammation in a range of human disease.
Neutrophilic inflammation is a prominent feature of COPD. Airway neutrophilia correlates with the rate of decline in lung function (12), and raised sputum neutrophil counts correlate with high-resolution computed tomography indicators of peripheral airway dysfunction in smokers (13). In addition, increased neutrophilic inflammation is characteristic of acute exacerbations of COPD (14). The vascular anatomy of the lung is unique in that the bronchi are supplied by the systemic circulation (bronchial arteries), whereas the alveolar capillary bed is fed by the low-pressure pulmonary circulation; migration of neutrophils into the alveoli occurs directly from the alveolar capillary bed rather than from the postcapillary venules as in the systemic circulation, and the selectin/integrin rolling/adhesion paradigm is not operative in this setting. Instead, the biophysical properties of the neutrophil and the unique microanatomy of the alveolar capillary bed exert a profound influence on neutrophil recruitment to the air spaces (15–17). It is tempting to speculate that differential recruitment of inflammatory cells to the airways and alveoli may underpin some of the phenotypic variants in COPD, but evidence to support this hypothesis is lacking. Exposure to cigarette smoke, pollutants, or infective agents has been shown to trigger the release of an array of chemoattractants from pulmonary epithelial cells and resident alveolar macrophages, promoting the recruitment of neutrophils and other inflammatory cells (18). This may lead to the establishment of a proinflammatory “vicious cycle,” with the products of neutrophil-mediated extracellular matrix (ECM) degradation, such as Pro-Gly-Pro (PGP) (19), functioning as chemoattractants. Cigarette smoke and other inflammatory stimuli promoted neutrophil sequestration in the lung directly by induction of cytoskeletal rearrangements, leading to reduced cellular deformability (20).
In addition to the well documented enhanced influx of neutrophils in COPD, it has become apparent that impaired efferocytic clearance of neutrophils may contribute to the dysregulated inflammation in this disease. Alveolar macrophages display defective phagocytic responses to a range of “prey” particles (21), including apoptotic cells (22), and cigarette smoke has been shown to impair efferocytosis of apoptotic neutrophils directly (23). TNF-α, a prominent cytokine in the COPD airway environment, has likewise been shown to impair macrophage uptake of apoptotic cells in vitro, and Borges and colleagues (24) showed that administration of TNF-α impaired the normally prompt removal of apoptotic cells from the lungs in a mouse model of neutrophilic inflammation (LPS-induced acute lung injury). As a result, the apoptotic cells underwent secondary necrosis, releasing proinflammatory components into the air spaces (24). Carbocysteine (a mucolytic used to promote sputum clearance) administered to mice promoted the ability of their alveolar macrophage to ingest apoptotic neutrophils (25). Drugs from the statin family (which have pleiotropic antiinflammatory properties) have been shown to promote neutrophil apoptosis (26) and to enhance the uptake of apoptotic cells (27). Clinical trials of statins in COPD are ongoing.
The presence of neutrophils in airway biopsies and in sputum samples represents single time points and does not necessarily reflect dynamic neutrophil flux. Our laboratory has recently reported increased lung accumulation of 99Tc-labeled neutrophils using sequential SPECT imaging, whereas whole-body counting demonstrated subsequent higher losses of 111ln-labeled neutrophils in patients who continued to smoke. These data are consistent with those obtained by Subramanian and colleagues (28) using quantitative positron emission tomography (28). Persson and Uller (29) have hypothesized that transepithelial migration of neutrophils across the airway wall is a protective mechanism to remove these potentially destructive cells while minimizing tissue injury, but Ginzberg and colleagues (30) have demonstrated that neutrophil transmigration induced epithelial cell apoptosis in a NE-dependent fashion. Further work by the same group (31) has suggested that the same process induces reparative signals in the injured epithelium, so the maintenance of epithelial integrity may depend on the magnitude and persistence of the neutrophil flux and the activation state of the transmigrating cells.
Thus, a combination of enhanced recruitment and failure of clearance may contribute to increased accumulation of neutrophils in the COPD lung and airway. Despite the increased presence of these phagocytes, bacterial infection contributes significantly to the pathophysiology of COPD. Failure to eliminate the resident pathogens suggests that the primary innate immune role of the neutrophil may be compromised in COPD.
The course of COPD is punctuated by recurrent episodes of clinical deterioration, referred to as “exacerbations.” Exacerbations are the leading cause of death in COPD and are associated with a decline in lung function and quality of life (32). Modern diagnostic techniques have revealed that the majority of exacerbations are triggered by viral or bacterial infection (recently reviewed by Sethi and colleagues [33]). Furthermore, bacterial pathogens can be isolated from the airways of patients with stable COPD, and a proportion of such individuals are persistently colonized with potentially pathogenic organisms such as Hemophilus spp (34) and Moraxella catarrhalis (35); such colonized individuals exhibit greater airways inflammation, more frequent exacerbations, and accelerated decline in lung function (36–39). Emerging data suggest that COPD airway microbiome is highly disordered even in apparently stable patients with COPD and that these abnormalities become more marked with disease severity (40–42); the significance of these observations is unclear.
Two recent trials of long-term treatment with macrolide antibiotics (43, 44) have concluded that this intervention reduced exacerbation frequency. The study by Seemungal and colleagues (43) (n = 109 patients) detected no difference in the frequency of positive sputum cultures during the trial, whereas the study by Albert and colleagues (44), which randomized 1,142 patients, showed a reduction in nasopharyngeal (but not lower airway) colonization with macrolide-sensitive organisms but a slight increase in macrolide-resistant isolates. There are no long-term data regarding outcomes such as disease progression and the impact of acquiring resistant organisms; until such data are available, the overall impact of such strategies remains unclear. It is also uncertain whether the effects of macrolides reflect their antibacterial activity or whether their well described immune-modulatory functions (recently reviewed in Kanoh and Rubin [45] and Idris and colleagues [46]) are of major relevance; of note, erythromycin reduced cigarette smoke–induced pulmonary inflammation in a sterile mouse model (47).
The phagocytic capacity of circulating monocytes and alveolar macrophages in COPD has been extensively studied, and there is abundant evidence that macrophage ingestion of bacteria is compromised in this disease (21). In contrast, few studies have addressed the ability of COPD neutrophils to ingest and kill bacterial prey, and the reported results have been inconsistent; depressed (48, 49) and unaffected (50) phagocytosis have been observed using isolated circulating COPD neutrophils in vitro. Cigarette smoke extract has been shown to reduce pulmonary clearance of Pseudomonas in a murine model of infection (51) and to impair neutrophil phagocytosis in vitro (52). The mechanisms underpinning any defect in the ability of neutrophils to ingest bacterial pathogens in COPD has been little studied; however, cleavage of opsonins and opsonic receptors by neutrophil proteases have been implicated. NE has been shown to cleave the complement fragment C3bi and the complement receptor CR1, an effect that could limit complement-mediated phagocytosis significantly (53). Toll-like receptor 2 expression (but not Toll-like receptor 4 expression) has been shown to be down-regulated on bronchoalveolar lavage (BAL) versus blood neutrophils in patients with COPD (54), potentially contributing to impaired phagocytic function. However, the number of subjects was limited, the receptor repertoire that was evaluated was restricted, and the mechanism for this down-regulation was not elucidated.
Once within the neutrophil phagosome, killing of ingested bacteria should occur by the assembly of the neutrophil NADPH oxidase and by the deployment of proteases and antibacterial proteins stored in neutrophil granules. The ability of neutrophils from patients with COPD to kill relevant pathogens has not been extensively studied, although McGovern and colleagues (55) reported that hypoxia impaired oxidant-dependent bacterial killing and promoted the survival of ingested Staphylococcus aureus. Ingestion of noncapsulated Hemophilus influenza (a common isolate from COPD sputum) by neutrophils has been shown to lead to death by necrosis of the engulfing phagocyte rather than bacterial killing, with the liberation of cytotoxic neutrophil granule contents (56). Although comparable studies with circulating neutrophils derived from COPD were not performed, neutrophils obtained from the sputum of patients with COPD infected with Hemophilus displayed a similar necrotic morphology. The mechanisms by which the ingested pathogen evaded neutrophil-mediated killing and evoked such profound neutrophil necrosis were not elucidated in this study and warrant further investigation.
Although receptor cleavage is likely to be an acquired defect secondary to the profoundly inflammatory milieu, a recent publication by Sapey and colleagues (57) has suggested that neutrophils from patients with COPD may have an intrinsic functional defect. These authors have shown that peripheral blood neutrophils from patients with COPD exhibited increased locomotor speed but greatly reduced chemotactic accuracy, irrespective of the severity of disease (Gold Stage 1–1V). This abnormality was corrected by inhibition of PI3 kinase, suggesting that enhanced generation of PIP3 or diminished metabolism of this second messenger by the phosphates PTEN and SHIP-1 may predispose to the impaired innate immune function in COPD and to the development of COPD.
Thus, COPD neutrophils seem to be recruited in a timely fashion in the event of infection but may fail to eliminate the invading pathogens. Infection in COPD does not result from “too little, too late” in terms of innate immunity; it results from a disordered host–pathogen interaction at the level of the phagocytic cells. It is not clear whether this abortive host–pathogen interaction is due to an intrinsic or acquired defect of neutrophil function, perhaps compounded by structural changes in the COPD airway, hypoxia, and dysfunction of other cellular components of the immune system. Further studies to ascertain the ability of COPD neutrophils to ingest and kill pathogenic bacteria would be of value, although such studies may be confounded by smoking status, the presence or absence of infection, and the treatments used for this disease. However, neutrophil dysfunction in COPD is not limited to failure to kill pathogens in the airway; several lines of evidence suggest that neutrophil-derived proteolytic mediators released extracellularly at sites of inflammation play an important role in the pathogenesis of a range of inflammatory diseases, including COPD. The remainder of this review focuses on the role of the neutrophil in causing tissue injury in the COPD pulmonary microenvironment.
Neutrophils possess four types of granules, formed sequentially during their differentiation in the bone marrow: primary (azurophil) granules, secondary (specific) granules, tertiary (gelatinase) granules, and secretory vesicles. Degranulation occurs in a hierarchical fashion, with the last formed (secretory vesicles) being the most readily mobilized (58). Azurophil granules, which contain NE, cathepsin, proteinase-3, and the majority of the histiotoxic proteases, require the most potent stimuli to induce significant release to the external milieu; however, neutrophils primed by exposure to cytokines (59) or by hypoxia (55) undergo greatly enhanced degranulation responses on stimulation. Hence, neutrophil priming enhances the destructive potential of the neutrophil granulocyte. Circulating neutrophils from patients with COPD have been shown to be primed (60), and this priming is particularly noted during exacerbations (61). The effects of cigarette smoke on neutrophil degranulation has been little studied, with early reports suggesting no effect (62) or a minor augmentation of elastase release by nicotine (63). Our own unpublished results suggest that cigarette smoke extract has little or no effect on neutrophil degranulation and does not enhance the ability of other stimuli to induce neutrophil exocytosis. Despite the apparent lack of effect of smoking on this process, there is abundant evidence that neutrophil degranulation releases a range of bioactive substances that contribute significantly to tissue injury in COPD.
Human neutrophil peptides, NE, IL-8, and matrix metalloproteinase (MMP)-9 were found to be elevated in COPD sputum samples and correlated with disease severity and with lung function decline at 2 years (64). NE and MMP-8 increased significantly (but transiently) in patients experiencing an exacerbation compared with stable COPD and healthy control subjects (65). The antimicrobial peptide neutrophil gelatinase-associated lipocalin has been found to be elevated in COPD BAL fluid (66), sputum (67), and serum (68), with serum neutrophil gelatinase-associated lipocalin positively correlated with exacerbation frequency and hypoxia (although not with GOLD stage). However, none of these markers is specific for COPD, and the quest to identify meaningful biomarkers in this debilitating condition is ongoing.
In addition to the documentation of the increased release of histotoxic neutrophil proteases in COPD, there is evidence that these enzymes contribute to tissue injury and hence pathogenesis, with the degradation products acting as proinflammatory stimuli in a feed-forward loop. PGP is a tripetide that is released when collagen is degraded by matrix metalloproteinases and acts as a neutrophil chemoattractant (19); PGP levels are elevated in COPD sputum and serum, and PGP generation by COPD sputum was blocked by inhibitors of MMP-1 and MMP-9 (69). Furthermore, Aα-Val360, a NE-specific cleavage product of fibrinogen (70), has been shown to be elevated in COPD and to correlate with markers of disease severity (71).
NE has an important role in neutrophil-mediated bacterial and fungal killing (72) but can also cause considerable damage when in contact with the ECM because it is capable of degrading almost every ECM component, including collagen, fibronectin, proteoglycans, heparin, and cross-linked fibrin (73). Stockley and colleagues (74) have shown that when neutrophils move into close proximity to connective tissue they degrade it; in part this may relate to the ability of the serine proteases to bind to the neutrophil surface after degranulation, where they seem to be resistant to protease inhibition (74). In addition, it has been documented that elastase molecules vastly outnumber the inhibitor molecules in the immediate pericellular zones after degranulation, allowing “quantum proteolysis” to occur (75). Another mechanism that is suggested is that upon degranulation serine proteases are able to bind to the cell surface (75, 76). When membrane bound, these enzymes have been shown to be remarkably resistant to the naturally occurring protease inhibitors (77). In addition to matrix destruction, NE induces direct epithelial damage (78), mucus gland hyperplasia (79), secretion of mucus (53), and reduced ciliary beat frequency (80); these changes may contribute to the ability of bacteria to invade and colonize the COPD airway documented above.
α1-Antitrypsin (A1AT) is a major endogenous inhibitor of serine proteases such as neutrophil elastase and proteinase-3. Although other antiproteases can inhibit elastase or proteinase-3, A1AT accounts for most of the antielastase at the alveolar level (81). Individuals with inherited A1AT deficiency display a dramatically increased susceptibility to chronic inflammatory conditions, in particular to cigarette smoke–induced COPD (82). A1AT deficiency underlies approximately 2% of all cases of COPD (83), a fact that provides strong support for the protease/antiprotease imbalance (84) hypothesis of COPD. Furthermore, replacement of A1AT in deficient individuals with established COPD has been shown to delay lung function decline (85).
Experimental emphysema in mice may be induced by the instillation of human neutrophil elastase and other elastolytic enzymes but not by nonelastolytic proteases (86, 87), and mice lacking NE are more resistant to cigarette smoke–induced emphysema (88). However, mice lacking macrophage elastase (MMP-12) are likewise protected (89), suggesting that a range of proteases may contribute to the pathogenesis of emphysema in the appropriate setting. Despite promising results in animal models of COPD (90), the oral elastase inhibitor AZD9668 showed no clinical efficacy and no effect of biomarkers of inflammation when administered for 3 months to patients with symptomatic COPD (91, 92). This observation may reflect the somewhat short duration of treatment but may also suggest that other neutrophil proteases contribute to COPD pathogenesis.
Myeloperoxidase (MPO), a 150-kD microbicidal hemoprotein, is present in the azurophilic granules together with NE (93), and its release is governed by the same mechanisms. MPO is capable of inducing oxidative stress in inflammatory states, particularly in the presence of cigarette smoke (94–96), and neutrophil MPO content is increased in smokers (97). There is also evidence that MPO, like NE, can directly up-regulate the inflammatory response; MPO internalized by endothelial cells leads to IL-6, IL-8, and ROS release, and MPO can induce TNF-α release from macrophages and activate neutrophils leading to increased degranulation (98, 99). 3-Chlorotyrosine, a specific product of MPO oxidative activity, was shown to be up-regulated in COPD sputum (100), suggesting a possible active role for MPO in smoking-related lung disease. Late intervention with the MPO inhibitor AZ1 in a guinea pig (cigarette smoke exposure) model of COPD stopped the progression of emphysema and small airway remodeling and was partially protective against pulmonary hypertension (101). This study suggests that the role of MPO in COPD warrants further investigation; however, as the authors note, positive findings in animal models of COPD have a disappointing record of failure in human disease (102).
MMP-9, also known as gelatinase B or neutrophil collagenase, is present in the gelatinase granules. Together with other MMPs, MMP-9 is capable of degrading major structural components of the extracellular matrix, including collagen, laminin, and gelatin, thereby facilitating neutrophil extravasation and migration (7, 75, 103) in controlled situations but with the potential to injure tissues if released indiscriminately. MMP-9 has been related to parenchymal destruction and lung function decline in subclinical and established emphysema (66, 104). Other studies have shown MMP-9 to be elevated in patients with COPD in comparison to healthy control subjects, and MMP-9 levels were found to correlate with increased neutrophilia and decreased function (105–107).
Because neutrophils do not synthesize the MMP-9 antiprotease TIMP-1, rapid and substantial release of this enzyme without restraint can occur (108). Vlahos and colleagues (109) demonstrated a dramatic up-regulation of MMP-9 in the BAL fluid of patients with COPD and noted a striking correlation with disease severity (21-fold increase in MMP-9 activity in GOLD II versus GOLD IV disease); there was a similar up-regulation of NE in the same samples, and it is noteworthy that NE can degrade TIMP-1, further promoting the action of MMP-9 (110). NE and MMP-9 release was insensitive to glucocorticoids but could be inhibited by targeting the PI3K pathway. Reminiscent of the data of Sapey and colleagues (57), this observation suggests that neutrophil-mediated tissue injury in COPD might be ameliorated by agents targeting the PI3K pathway, although this approach might also confer increased susceptibility to infection by compromising other aspects of immune cell function.
There is abundant evidence that a range of neutrophil-derived proteases contribute to the pathogenesis of COPD and to the maintenance of the inflammatory phenotype in this condition. Therapeutic strategies directed at this aspect of aberrant neutrophil function in COPD may need to target a broad spectrum rather than a single protease and could be more beneficial if administered early in the course of the disease before major irreversible tissue destruction has occurred.
Chronic and progressive hypoxia is one of the fundamental features of severe COPD and the degree of hypoxia correlates with markers of inflammation (111, 112). Profound local tissue hypoxia may occur in the absence of systemic hypoxia, particularly in the context of infection or inflammation. Detection of the transcription factor hypoxia-inducible factor (HIF)1α, together with membrane-associated carbonic anhydrase, is a powerful indicator of cellular hypoxia (113, 114). Polosukhin and colleagues (115) showed that both markers were expressed in the bronchial epithelial cells in COPD airways, particularly in association with airway remodeling. HIF-1α expression was also associated with areas of goblet cell hyperplasia in the airway epithelium of patients with COPD (116). Thus, in COPD, neutrophils and other inflammatory cells may be exposed to a range of oxygen tensions that include quite profound hypoxia. To function effectively in this setting, neutrophils must be able to respond to changing oxygen levels, and this is achieved principally by HIF-mediated signaling. Stabilization of HIF-1α by pharmacological or genetic manipulation has been shown to delay the resolution of neutrophilic inflammation in a zebrafish model (117), confirming the potential of hypoxia and hypoxia signaling to modulate inflammatory responses in vivo.
Hypoxia has been shown to have a profound effect on neutrophil function. Neutrophils exposed to physiological levels of hypoxia in vitro exhibited dramatically prolonged survival (118), and this was found to be a key component in the prolonged inflammatory response seen by Elks and colleagues (117). Increasing the rate of neutrophil apoptosis has been shown to accelerate the resolution of inflammation in a number of inflammatory models (119). In addition, sustained hypoxia was shown to impair the oxidative burst and oxidase-dependent bacterial killing and to promote bacterial survival (55). Hypoxia and cytokine exposure led to a highly significant increase in extracellular NE release. Hypoxia may also sensitize tissues to NE-mediated cellular damage (120) and may suppress elastin repair (121), compounding the injurious potential of the enhanced NE release. Together, these changes constitute a highly destructive neutrophil phenotype and recapitulate the defects seen in the broader picture of COPD: impaired innate immune capability and enhanced capacity for tissue destruction (“too much, too soon”).
COPD is no longer regarded as being a disease affecting only the lungs but as a complex, heterogeneous, and systemic disorder manifest in an aging population. Extrapulmonary comorbidities complicate the management and influence the prognosis of patients with COPD, but the drivers of these systemic manifestations are unclear. There is increasing evidence that chronic inflammation, potentially driven by the combination of smoking, airway colonization, and oxidative stress, is a key factor in the evolution of COPD. A number of recent articles have described the local and systemic biomarkers of inflammation associated with COPD (122–124), and systemic biomarkers of neutrophilic inflammation have been correlated with disease severity (125). Circulating neutrophils have been noted to be primed for enhanced responsiveness (degranulation and oxidative burst) in stable disease (60) and during exacerbations (61, 126), perhaps due to exposure to circulating cytokines. Priming is regarded as a prerequisite for neutrophil-mediated tissue injury, and primed neutrophils are more likely to sequester in the pulmonary vasculature in animal models (127) and in humans (128). Circulating neutrophils also exhibit prolonged survival during COPD exacerbations (126, 129), although analyses of sputum neutrophil lifespan have yielded conflicting results (130–132).
Despite the evidence for systemic activation of neutrophils in COPD, their potential role in mediating the systemic features of this disease has been little studied. One exception to this is the recent study by Menon and colleagues (133) demonstrating that neutrophil numbers were significantly elevated in quadriceps muscle biopsies of patients with COPD, with further increases in the inflammatory infiltrate seen after acute resistance exercise. Further studies to elucidate the role of neutrophils in the systemic inflammatory complications of COPD would be a welcome addition to our knowledge of this debilitating disease.
Exciting new discoveries place the neutrophil center stage in the pathogenesis of COPD (Figure 1). Cigarette smoke, pathogenic organisms, and other noxious stimuli elicit a florid neutrophil inflammation, which is compounded by ECM destruction with the generation of more sustained recruitment signals. Cytokines and hypoxia synergize to enhance the release of proteases, leading to cellular and ECM damage and perpetuating the vicious cycle. Hypoxia and intrinsic or acquired defects in neutrophil function, plus damage to the local microenvironment, compromise the ability of the host to clear pathogenic organisms, further contributing to the proinflammatory environment in COPD. These advances have promoted renewed interest in the neutrophil as a therapeutic target in COPD, and a range of antiinflammatory agents targeting neutrophil recruitment, activation, protease release and function, and survival are in phase 1 and phase 2 clinical trials.
Figure 1. Neutrophil function in healthy and chronic obstructive pulmonary disease (COPD) airways. Healthy airways are protected from infection by the innate immune system; bacteria are cleared by the mucociliary escalator, by the action of antibacterial peptides, and by resident phagocytes. Pathogens that evade these first-line defenses are ingested and killed by recruited neutrophils, which then undergo apoptosis and efferocytosis, controlling infection and limiting inflammation. In the COPD airway, cigarette smoke exposure may damage local defenses, with impairment of the mucociliary system and the phagocytic capacity of alveolar macrophages. Cigarette smoke and pathogens trigger the release of chemoattractants, promoting the recruitment of neutrophils and other inflammatory cells. Cigarette smoke may also impair the efferocytosis of apoptotic neutrophils, allowing them to undergo secondary necrosis and hence creating further proinflammatory signals. Although there is increased neutrophil infiltration, local or systemic hypoxia impair bacterial killing and increase the release of proteases, resulting in further mucociliary dysfunction and cellular and tissue injury. The proinflammatory state is perpetuated by increased neutrophil lifespan secondary to hypoxia and inflammatory cytokines. ECM = extracellular matrix.
[More] [Minimize]| 1. | WHO. The top 10 causes of death [internet]. Available from: http://www.who.int/mediacentre/factsheets/fs310/en/index.html (accessed January 2, 2013). |
| 2. | Franklin W, Lowell FC, Michelson AL, Schiller IW. Chronic obstructive pulmonary emphysema: a disease of smokers. Ann Intern Med 1956;45:268–274. |
| 3. | Fletcher C, Peto R. The natural history of chronic airflow obstruction. BMJ 1977;1:1645–1648. |
| 4. | Lundbäck B, Lindberg A, Lindström M, Rönmark E, Jonsson AC, Jönsson E, Larsson LG, Andersson S, Sandström T, Larsson K; Obstructive Lung Disease in Northern Sweden Studies. Not 15 but 50% of smokers develop COPD? Report from the Obstructive Lung Disease in Northern Sweden Studies. Respir Med 2003;97:115–122. |
| 5. | Fischer BM, Pavlisko E, Voynow JA. Pathogenic triad in COPD: oxidative stress, protease-antiprotease imbalance, and inflammation. Int J Chron Obstruct Pulmon Dis 2011;6:413–421. |
| 6. | Provinciali M, Cardelli M, Marchegiani F. Inflammation, chronic obstructive pulmonary disease and aging. Curr Opin Pulm Med 2011;17:S3–S10. |
| 7. | Borregaard N, Cowland JB. Granules of the human neutrophilic polymorphonuclear leukocyte. Blood 1997;89:3503–3521. |
| 8. | Ludwig PW, Schwartz BA, Hoidal JR, Niewoehner DE. Cigarette smoking causes accumulation of polymorphonuclear leukocytes in alveolar septum. Am Rev Respir Dis 1985;131:828–830. |
| 9. | Martin TR, Raghu G, Maunder RJ, Springmeyer SC. The effects of chronic bronchitis and chronic air-flow obstruction on lung cell populations recovered by bronchoalveolar lavage. Am Rev Respir Dis 1985;132:254–260. |
| 10. | Pesci A, Majori M, Cuomo A, Borciani N, Bertacco S, Cacciani G, Gabrielli M. Neutrophils infiltrating bronchial epithelium in chronic obstructive pulmonary disease. Respir Med 1998;92:863–870. |
| 11. | Pilette C, Colinet B, Kiss R, André S, Kaltner H, Gabius HJ, Delos M, Vaerman JP, Decramer M, Sibille Y. Increased galectin-3 expression and intra-epithelial neutrophils in small airways in severe COPD. Eur Respir J 2007;29:914–922. |
| 12. | Stănescu D, Sanna A, Veriter C, Kostianev S, Calcagni PG, Fabbri LM, Maestrelli P. Airways obstruction, chronic expectoration, and rapid decline of FEV1 in smokers are associated with increased levels of sputum neutrophils. Thorax 1996;51:267–271. |
| 13. | O’Donnell RA, Peebles C, Ward JA, Daraker A, Angco G, Broberg P, Pierrou S, Lund J, Holgate ST, Davies DE, et al. Relationship between peripheral airway dysfunction, airway obstruction, and neutrophilic inflammation in COPD. Thorax 2004;59:837–842. |
| 14. | Gompertz S, O’Brien C, Bayley DL, Hill SL, Stockley RA. Changes in bronchial inflammation during acute exacerbations of chronic bronchitis. Eur Respir J 2001;17:1112–1119. |
| 15. | Burns AR, Smith CW, Walker DC. Unique structural features that influence neutrophil emigration into the lung. Physiol Rev 2003;83:309–336. |
| 16. | Gane J, Stockley R. Mechanisms of neutrophil transmigration across the vascular endothelium in COPD. Thorax 2012;67:553–561. |
| 17. | Worthen GS, Schwab B III, Elson EL, Downey GP. Mechanics of stimulated neutrophils: cell stiffening induces retention in capillaries. Science 1989;245:183–186. |
| 18. | Barnes PJ. The cytokine network in chronic obstructive pulmonary disease. Am J Respir Cell Mol Biol 2009;41:631–638. |
| 19. | Weathington NM, et al. A novel peptide CXCR ligand derived from extracellular matrix degradation during airway inflammation. Nat Med 2006;12:317–323. |
| 20. | Drost EM, Selby C, Bridgeman MME, Macnee W. Decreased leukocyte deformability after acute cigarette smoking in humans. Am J Respir Crit Care Med 1993;148:1277–1283. |
| 21. | Donnelly LE, Barnes PJ. Defective phagocytosis in airways disease. Chest 2012;141:1055–1062. |
| 22. | Hodge S, Hodge G, Scicchitano R, Reynolds PN, Holmes M. Alveolar macrophages from subjects with chronic obstructive pulmonary disease are deficient in their ability to phagocytose apoptotic airway epithelial cells. Immunol Cell Biol 2003;81:289–296. |
| 23. | Richens TR, Linderman DJ, Horstmann SA, Lambert C, Xiao YQ, Keith RL, Boé DM, Morimoto K, Bowler RP, Day BJ, et al. Cigarette smoke impairs clearance of apoptotic cells through oxidant-dependent activation of RhoA. Am J Respir Crit Care Med 2009;179:1011–1021. |
| 24. | Borges VM, Vandivier RW, McPhillips KA, Kench JA, Morimoto K, Groshong SD, Richens TR, Graham BB, Muldrow AM, Van Heule L, et al. TNFα inhibits apoptotic cell clearance in the lung, exacerbating acute inflammation. Am J Physiol Lung Cell Mol Physiol 2009;297:L586–L595. |
| 25. | Inoue M, Ishibashi Y, Nogawa H, Yasue T. Carbocisteine promotes phagocytosis of apoptotic cells by alveolar macrophages. Eur J Pharmacol 2012;677:173–179. |
| 26. | Chello M, Anselmi A, Spadaccio C, Patti G, Goffredo C, Di Sciascio G, Covino E. Simvastatin increases neutrophil apoptosis and reduces inflammatory reaction after coronary surgery. Ann Thorac Surg 2007;83:1374–1380. |
| 27. | Morimoto K, Janssen WJ, Fessler MB, McPhillips KA, Borges VM, Bowler RP, Xiao YQ, Kench JA, Henson PM, Vandivier RW. Lovastatin enhances clearance of apoptotic cells (efferocytosis) with implications for chronic obstructive pulmonary disease. J Immunol 2006;176:7657–7665. |
| 28. | Subramanian DR, Jenkins L, Edgar R, Quraishi N, Stockley RA, Parr DG. Assessment of pulmonary neutrophilic inflammation in emphysema by quantitative positron emission tomography. Am J Respir Crit Care Med 2012;186:1125–1132. |
| 29. | Persson C, Uller L. Transepithelial exit of leucocytes: inflicting, reflecting or resolving airway inflammation? Thorax 2010;65:1111–1115. |
| 30. | Ginzberg HH, Cherapanov V, Dong Q, Cantin A, McCulloch CA, Shannon PT, Downey GP. Neutrophil-mediated epithelial injury during transmigration: role of elastase. Am J Physiol Gastrointest Liver Physiol 2001;281:G705–G717. |
| 31. | Zemans RL, Briones N, Campbell M, McClendon J, Young SK, Suzuki T, Yang IV, De Langhe S, Reynolds SD, Mason RJ, et al. Neutrophil transmigration triggers repair of the lung epithelium via beta-catenin signaling. Proc Natl Acad Sci USA 2011;108:15990–15995. |
| 32. | Donaldson GC, Seemungal TAR, Bhowmik A, Wedzicha JA. Relationship between exacerbation frequency and lung function decline in chronic obstructive pulmonary disease. Thorax 2002;57:847–852. |
| 33. | Sethi S. Molecular diagnosis of respiratory tract infection in acute exacerbations of chronic obstructive pulmonary disease. Clin Infect Dis 2011;52:S290–S295. |
| 34. | Murphy TF, Brauer AL, Schiffmacher AT, Sethi S. Persistent colonization by Haemophilus influenzae in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2004;170:266–272. |
| 35. | Parameswaran GI, Wrona CT, Murphy TF, Sethi S. Moraxella catarrhalis acquisition, airway inflammation and protease-antiprotease balance in chronic obstructive pulmonary disease. BMC Infect Dis 2009;9:178. |
| 36. | Patel IS, Seemungal TA, Wilks M, Lloyd-Owen SJ, Donaldson GC, Wedzicha JA. Relationship between bacterial colonisation and the frequency, character, and severity of COPD exacerbations. Thorax 2002;57:759–764. |
| 37. | Wilkinson TMA, Patel IS, Wilks M, Donaldson GC, Wedzicha JA. Airway bacterial load and FEV1 decline in patients with chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2003;167:1090–1095. |
| 38. | Banerjee D, Khair OA, Honeybourne D. Impact of sputum bacteria on airway inflammation and health status in clinical stable COPD. Eur Respir J 2004;23:685–691. |
| 39. | Hill AT, Campbell EJ, Hill SL, Bayley DL, Stockley RA. Association between airway bacterial load and markers of airway inflammation in patients with stable chronic bronchitis. Am J Med 2000;109:288–295. |
| 40. | Cabrera-Rubio R, Garcia-Núñez M, Setó L, Antó JM, Moya A, Monsó E, Mira A. Microbiome diversity in the bronchial tract of patients with chronic obstructive pulmonary disease. J Clin Microbiol 2012;50:3562–3568. |
| 41. | Han MK, Huang YJ, Lipuma JJ, Boushey HA, Boucher RC, Cookson WO, Curtis JL, Erb-Downward J, Lynch SV, Sethi S, et al. Significance of the microbiome in obstructive lung disease. Thorax 2012;67:456–463. |
| 42. | Sze MA, Dimitriu PA, Hayashi S, Elliott WM, McDonough JE, Gosselink JV, Cooper J, Sin DD, Mohn WW, Hogg JC. The lung tissue microbiome in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2012;185:1073–1080. |
| 43. | Seemungal TAR, Wilkinson TM, Hurst JR, Perera WR, Sapsford RJ, Wedzicha JA. Long-term erythromycin therapy is associated with decreased chronic obstructive pulmonary disease exacerbations. Am J Respir Crit Care Med 2008;178:1139–1147. |
| 44. | Albert RK, Connett J, Woodruff PG. Azithromycin and the risk of cardiovascular death. N Engl J Med 2012;367:773–774, author reply 775. |
| 45. | Kanoh S, Rubin BK. Mechanisms of action and clinical application of macrolides as immunomodulatory medications. Clin Microbiol Rev 2010;23:590–615. |
| 46. | Idris SF, Chilvers ER, Haworth C, McKeon D, Condliffe AM. Azithromycin therapy for neutrophilic airways disease: myth or magic? Thorax 2009;64:186–189. |
| 47. | Mikura S, Wada H, Higaki M, Yasutake T, Ishii H, Kamiya S, Goto H. Erythromycin prevents the pulmonary inflammation induced by exposure to cigarette smoke. Transl Res 2011;158:30–37. |
| 48. | Fietta A, Bersani C, De Rose V, Grassi FA, Mangiarotti P, Uccelli M, Grassi C. Evaluation of systemic host defense mechanisms in chronic bronchitis. Respiration 1988;53:37–43. |
| 49. | Prieto A, Reyes E, Bernstein ED, Martinez B, Monserrat J, Izquierdo JL, Callol L, de LUCAS P, Alvarez-Sala R, Alvarez-Sala JL, et al. Defective natural killer and phagocytic activities in chronic obstructive pulmonary disease are restored by glycophosphopeptical (inmunoferón). Am J Respir Crit Care Med 2001;163:1578–1583. |
| 50. | Müns G, Rubinstein I, Singer P. Phagocytosis and oxidative burst of granulocytes in the upper respiratory tract in chronic and acute inflammation. J Otolaryngol 1995;24:105–110. |
| 51. | Drannik AG, Pouladi MA, Robbins CS, Goncharova SI, Kianpour S, Stämpfli MR. Impact of cigarette smoke on clearance and inflammation after Pseudomonas aeruginosa infection. Am J Respir Crit Care Med 2004;170:1164–1171. |
| 52. | Stringer KA, Tobias M, O’Neill HC, Franklin CC. Cigarette smoke extract-induced suppression of caspase-3-like activity impairs human neutrophil phagocytosis. Am J Physiol Lung Cell Mol Physiol 2007;292:L1572–L1579. |
| 53. | Tosi MF, Zakem H, Berger M. Neutrophil elastase cleaves C3bi on opsonized pseudomonas as well as CR1 on neutrophils to create a functionally important opsonin receptor mismatch. J Clin Invest 1990;86:300–308. |
| 54. | Von Scheele I, Larsson K, Dahlén B, Billing B, Skedinger M, Lantz AS, Palmberg L. Toll-like receptor expression in smokers with and without COPD. Respir Med 2011;105:1222–1230. |
| 55. | McGovern NN, Cowburn AS, Porter L, Walmsley SR, Summers C, Thompson AA, Anwar S, Willcocks LC, Whyte MK, Condliffe AM, Chilvers ER. Hypoxia selectively inhibits respiratory burst activity and killing of Staphylococcus aureus in human neutrophils. J Immunol 2011;186:453–463. |
| 56. | Naylor EJ, Bakstad D, Biffen M, Thong B, Calverley P, Scott S, Hart CA, Moots RJ, Edwards SW. Haemophilus influenzae induces neutrophil necrosis: a role in chronic obstructive pulmonary disease? Am J Respir Cell Mol Biol 2007;37:135–143. |
| 57. | Sapey E, Stockley JA, Greenwood H, Ahmad A, Bayley D, Lord JM, Insall RH, Stockley RA. Behavioral and structural differences in migrating peripheral neutrophils from patients with chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2011;183:1176–1186. |
| 58. | Sengeløv H, Kjeldsen L, Borregaard N. Control of exocytosis in early neutrophil activation. J Immunol 1993;150:1535–1543. |
| 59. | Cadwallader KA, Uddin M, Condliffe AM, Cowburn AS, White JF, Skepper JN, Ktistakis NT, Chilvers ER. Effect of priming on activation and localization of phospholipase D-1 in human neutrophils. Eur J Biochem 2004;271:2755–2764. |
| 60. | Koenderman L, Kanters D, Maesen B, Raaijmakers J, Lammers JW, de Kruif J, Logtenberg T. Monitoring of neutrophil priming in whole blood by antibodies isolated from a synthetic phage antibody library. J Leukoc Biol 2000;68:58–64. |
| 61. | Oudijk E-JD, Gerritsen WB, Nijhuis EH, Kanters D, Maesen BL, Lammers JW, Koenderman L. Expression of priming-associated cellular markers on neutrophils during an exacerbation of COPD. Respir Med 2006;100:1791–1799. |
| 62. | Totti N III, McCusker KT, Campbell EJ, Griffin GL, Senior RM. Nicotine is chemotactic for neutrophils and enhances neutrophil responsiveness to chemotactic peptides. Science 1984;223:169–171. |
| 63. | Seow WK, Thong YH, Nelson RD, MacFarlane GD, Herzberg MC. Nicotine-induced release of elastase and eicosanoids by human neutrophils. Inflammation 1994;18:119–127. |
| 64. | Paone G, Conti V, Vestri A, Leone A, Puglisi G, Benassi F, Brunetti G, Schmid G, Cammarella I, Terzano C. Analysis of sputum markers in the evaluation of lung inflammation and functional impairment in symptomatic smokers and COPD patients. Dis Markers 2011;31:91–100. |
| 65. | Ilumets H, Rytilä PH, Sovijärvi AR, Tervahartiala T, Myllärniemi M, Sorsa TA, Kinnula VL. Transient elevation of neutrophil proteinases in induced sputum during COPD exacerbation. Scand J Clin Lab Invest 2008;68:618–623. |
| 66. | Betsuyaku T, Nishimura M, Takeyabu K, Tanino M, Venge P, Xu S, Kawakami Y. Neutrophil granule proteins in bronchoalveolar lavage fluid from subjects with subclinical emphysema. Am J Respir Crit Care Med 1999;159:1985–1991. |
| 67. | Keatings VM, Barnes PJ. Granulocyte activation markers in induced sputum: comparison between chronic obstructive pulmonary disease, asthma, and normal subjects. Am J Respir Crit Care Med 1997;155:449–453. |
| 68. | Eagan TM, Damås JK, Ueland T, Voll-Aanerud M, Mollnes TE, Hardie JA, Bakke PS, Aukrust P. Neutrophil gelatinase-associated lipocalin: a biomarker in COPD. Chest 2010;138:888–895. |
| 69. | O’Reilly P, Jackson PL, Noerager B, Parker S, Dransfield M, Gaggar A, Blalock JE. N-alpha-PGP and PGP, potential biomarkers and therapeutic targets for COPD. Respir Res 2009;10:38. |
| 70. | Carter RI, Mumford RA, Treonze KM, Finke PE, Davies P, Si Q, Humes JL, Dirksen A, Piitulainen E, Ahmad A, et al. The fibrinogen cleavage product Aα-Val360, a specific marker of neutrophil elastase activity in vivo. Thorax 2011;66:686–691. |
| 71. | Carter RI, Ungurs MJ, Mumford RA, Stockley RAA. α-Val360: a marker of neutrophil elastase and COPD disease activity. Eur Respir J 2012013;41:31–38. |
| 72. | Tkalcevic J, Novelli M, Phylactides M, Iredale JP, Segal AW, Roes J. Impaired immunity and enhanced resistance to endotoxin in the absence of neutrophil elastase and cathepsin G. Immunity 2000;12:201–210. |
| 73. | Travis J. Structure, function, and control of neutrophil proteinases. Am J Med 1988;84:37–42. |
| 74. | Stockley RA. Neutrophils and protease/antiprotease imbalance. Am J Respir Crit Care Med 1999;160:S49–S52. |
| 75. | Owen CA, Campbell MA, Sannes PL, Boukedes SS, Campbell EJ. Cell surface-bound elastase and cathepsin G on human neutrophils: a novel, non-oxidative mechanism by which neutrophils focus and preserve catalytic activity of serine proteinases. J Cell Biol 1995;131:775–789. |
| 76. | Pham CTN. Neutrophil serine proteases: specific regulators of inflammation. Nat Rev Immunol 2006;6:541–550. |
| 77. | Campbell EJ, Campbell MA, Boukedes SS, Owen CA. Quantum proteolysis by neutrophils: implications for pulmonary emphysema in alpha 1-antitrypsin deficiency. J Clin Invest 1999;104:337–344. |
| 78. | Amitani R, Wilson R, Rutman A, Read R, Ward C, Burnett D, Stockley RA, Cole PJ. Effects of human neutrophil elastase and Pseudomonas aeruginosa proteinases on human respiratory epithelium. Am J Respir Cell Mol Biol 1991;4:26–32. |
| 79. | Lucey EC, Stone PJ, Breuer R, Christensen TG, Calore JD, Catanese A, Franzblau C, Snider GL. Effect of combined human neutrophil cathepsin G and elastase on induction of secretory cell metaplasia and emphysema in hamsters, with in vitro observations on elastolysis by these enzymes. Am Rev Respir Dis 1985;132:362–366. |
| 80. | Smallman LA, Hill SL, Stockley RA. Reduction of ciliary beat frequency in vitro by sputum from patients with bronchiectasis: a serine proteinase effect. Thorax 1984;39:663–667. |
| 81. | Gadek JE, Fells GA, Zimmerman RL, Rennard SI, Crystal RG. Antielastases of the human alveolar structures: implications for the protease-antiprotease theory of emphysema. J Clin Invest 1981;68:889–898. |
| 82. | Talamo RC, Blennerhassett JB, Austen KF. Familial emphysema and alpha-1-antitrypsin deficiency. N Engl J Med 1966;275:1301–1304. |
| 83. | Lieberman J, Winter B, Sastre A. Alpha 1-antitrypsin Pi-types in 965 COPD patients. Chest 1986;89:370–373. |
| 84. | Lieberman J. Elastase, collagenase, emphysema, and alpha1-antitrypsin deficiency. Chest 1976;70:62–67. |
| 85. | Chapman KR, Stockley RA, Dawkins C, Wilkes MM, Navickis RJ. Augmentation therapy for alpha1 antitrypsin deficiency: a meta-analysis. COPD 2009;6:177–184. |
| 86. | Lungarella G, Cavarra E, Lucattelli M, Martorana PA. The dual role of neutrophil elastase in lung destruction and repair. Int J Biochem Cell Biol 2008;40:1287–1296. |
| 87. | Korkmaz B, Horwitz MS, Jenne DE, Gauthier F. Neutrophil elastase, proteinase 3, and cathepsin G as therapeutic targets in human diseases. Pharmacol Rev 2010;62:726–759. |
| 88. | Shapiro SD, Goldstein NM, Houghton AM, Kobayashi DK, Kelley D, Belaaouaj A. Neutrophil elastase contributes to cigarette smoke-induced emphysema in mice. Am J Pathol 2003;163:2329–2335. |
| 89. | Hautamaki RD, Kobayashi DK, Senior RM, Shapiro SD. Requirement for macrophage elastase for cigarette smoke-induced emphysema in mice. Science 1997;277:2002–2004. |
| 90. | Stevens T, Ekholm K, Gränse M, Lindahl M, Kozma V, Jungar C, Ottosson T, Falk-Håkansson H, Churg A, Wright JL, et al. AZD9668: pharmacological characterization of a novel oral inhibitor of neutrophil elastase. J Pharmacol Exp Ther 2011;339:313–320. |
| 91. | Kuna P, Jenkins M, O’Brien CD, Fahy WA. AZD9668, a neutrophil elastase inhibitor, plus ongoing budesonide/formoterol in patients with COPD. Respir Med 2012;106:531–539. |
| 92. | Vogelmeier C, Aquino TO, O’Brien CD, Perrett J, Gunawardena KA. A randomised, placebo-controlled, dose-finding study of AZD9668, an oral inhibitor of neutrophil elastase, in patients with chronic obstructive pulmonary disease treated with tiotropium. COPD 2012;9:111–120. |
| 93. | Faurschou M, Sørensen OE, Johnsen AH, Askaa J, Borregaard N. Defensin-rich granules of human neutrophils: characterization of secretory properties. Biochim Biophys Acta 2002;1591:29–35. |
| 94. | Church DF, Pryor WA. Free-radical chemistry of cigarette smoke and its toxicological implications. Environ Health Perspect 1985;64:111–126. |
| 95. | Nakayama T, Church DF, Pryor WA. Quantitative analysis of the hydrogen peroxide formed in aqueous cigarette tar extracts. Free Radic Biol Med 1989;7:9–15. |
| 96. | Heinecke JW. Tyrosyl radical production by myeloperoxidase: a phagocyte pathway for lipid peroxidation and dityrosine cross-linking of proteins. Toxicology 2002;177:11–22. |
| 97. | Bridges RB, Fu MC, Rehm SR. Increased neutrophil myeloperoxidase activity associated with cigarette smoking. Eur J Respir Dis 1985;67:84–93. |
| 98. | Lefkowitz DL, Roberts E, Grattendick K, Schwab C, Stuart R, Lincoln J, Allen RC, Moguilevsky N, Bollen A, Lefkowitz SS. The endothelium and cytokine secretion: the role of peroxidases as immunoregulators. Cell Immunol 2000;202:23–30. |
| 99. | Yang JJ, Preston GA, Pendergraft WF, Segelmark M, Heeringa P, Hogan SL, Jennette JC, Falk RJ. Internalization of proteinase 3 is concomitant with endothelial cell apoptosis and internalization of myeloperoxidase with generation of intracellular oxidants. Am J Pathol 2001;158:581–592. |
| 100. | O’Donnell C, Newbold P, White P, Thong B, Stone H, Stockley RA. 3-Chlorotyrosine in sputum of COPD patients: relationship with airway inflammation. COPD 2010;7:411–417. |
| 101. | Churg A, Marshall CV, Sin DD, Bolton S, Zhou S, Thain K, Cadogan EB, Maltby J, Soars MG, Mallinder PR, et al. Late intervention with a myeloperoxidase inhibitor stops progression of experimental chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2012;185:34–43. |
| 102. | Churg A, Sin DD, Wright JL. Everything prevents emphysema: are animal models of cigarette smoke-induced chronic obstructive pulmonary disease any use? Am J Respir Cell Mol Biol 2011;45:1111–1115. |
| 103. | Kang T, Yi J, Guo A, Wang X, Overall CM, Jiang W, Elde R, Borregaard N, Pei D. Subcellular distribution and cytokine- and chemokine-regulated secretion of leukolysin/MT6-MMP/MMP-25 in neutrophils. J Biol Chem 2001;276:21960–21968. |
| 104. | Finlay GA, Russell KJ, McMahon KJ, D'arcy EM, Masterson JB, FitzGerald MX, O’Connor CM. Elevated levels of matrix metalloproteinases in bronchoalveolar lavage fluid of emphysematous patients. Thorax 1997;52:502–506. |
| 105. | Mercer P, Shute JK, Bhowmik A, Donaldson GC, Wedzicha JA, Warner JA. MMP-9, TIMP-1 and inflammatory cells in sputum from COPD patients during exacerbation. Respir Res 2005;6:151. |
| 106. | Vignola AM, Riccobono L, Mirabella A, Profita M, Chanez P, Bellia V, Mautino G, D’accardi P, Bousquet J, Bonsignore G. Sputum metalloproteinase-9/tissue inhibitor of metalloproteinase-1 ratio correlates with airflow obstruction in asthma and chronic bronchitis. Am J Respir Crit Care Med 1998;158:1945–1950. |
| 107. | Vernooy JHJ, Lindeman JHN, Jacobs JA, Hanemaaijer R, Wouters EFM. Increased activity of matrix metalloproteinase-8 and matrix metalloproteinase-9 in induced sputum from patients with COPD. Chest 2004;126:1802–1810. |
| 108. | Masure S, Proost P, Van Damme J, Opdenakker G. Purification and identification of 91-kDa neutrophil gelatinase: release by the activating peptide interleukin-8. Eur J Biochem 1991;198:391–398. |
| 109. | Vlahos R, Wark PAB, Anderson GP, Bozinovski S. Glucocorticosteroids differentially regulate MMP-9 and neutrophil elastase in COPD. PLoS ONE 2012;7:e33277. |
| 110. | Itoh Y, Nagase H. Preferential inactivation of tissue inhibitor of metalloproteinases-1 that is bound to the precursor of matrix metalloproteinase 9 (progelatinase B) by human neutrophil elastase. J Biol Chem 1995;270:16518–16521. |
| 111. | Baldi S, Pinna GD, Mombaruzzo P, Biglieri M, De Martini A, Palange P. C-reactive protein correlates with tissue oxygen availability in patients with stable COPD. Int J Chron Obstruct Pulmon Dis 2008;3:745–751. |
| 112. | Sabit R, Thomas P, Shale DJ, Collins P, Linnane SJ. The effects of hypoxia on markers of coagulation and systemic inflammation in patients with COPD. Chest 2010;138:47–51. |
| 113. | Haugland HK, Vukovic V, Pintilie M, Fyles AW, Milosevic M, Hill RP, Hedley DW. Expression of hypoxia-inducible factor-1alpha in cervical carcinomas: correlation with tumor oxygenation. Int J Radiat Oncol Biol Phys 2002;53:854–861. |
| 114. | Williams KJ, Parker CA, Stratford IJ. Exogenous and endogenous markers of tumour oxygenation status: definitive markers of tumour hypoxia? Adv Exp Med Biol 2005;566:285–294. |
| 115. | Polosukhin VV, Lawson WE, Milstone AP, Egunova SM, Kulipanov AG, Tchuvakin SG, Massion PP, Blackwell TS. Association of progressive structural changes in the bronchial epithelium with subepithelial fibrous remodeling: a potential role for hypoxia. Virchows Arch 2007;451:793–803. |
| 116. | Polosukhin VV, Cates JM, Lawson WE, Milstone AP, Matafonov AG, Massion PP, Lee JW, Randell SH, Blackwell TS. Hypoxia-inducible factor-1 signalling promotes goblet cell hyperplasia in airway epithelium. J Pathol 2011;224:203–211. |
| 117. | Elks PM, van Eeden FJ, Dixon G, Wang X, Reyes-Aldasoro CC, Ingham PW, Whyte MK, Walmsley SR, Renshaw SA. Activation of hypoxia-inducible factor-1α (Hif-1α) delays inflammation resolution by reducing neutrophil apoptosis and reverse migration in a zebrafish inflammation model. Blood 2011;118:712–722. |
| 118. | Hannah S, Mecklenburgh K, Rahman I, Bellingan GJ, Greening A, Haslett C, Chilvers ER. Hypoxia prolongs neutrophil survival in vitro. FEBS Lett 1995;372:233–237. |
| 119. | Heasman SJ, Giles KM, Ward C, Rossi AG, Haslett C, Dransfield I. Glucocorticoid-mediated regulation of granulocyte apoptosis and macrophage phagocytosis of apoptotic cells: implications for the resolution of inflammation. J Endocrinol 2003;178:29–36. |
| 120. | Sparkenbaugh EM, Ganey PE, Roth RA. Hypoxia sensitization of hepatocytes to neutrophil elastase-mediated cell death depends on MAPKs and HIF-1α. Am J Physiol Gastrointest Liver Physiol 2012;302:G748–G757. |
| 121. | Berk JL, Hatch CA, Morris SM, Stone PJ, Goldstein RH. Hypoxia suppresses elastin repair by rat lung fibroblasts. Am J Physiol Lung Cell Mol Physiol 2005;289:L931–L936. |
| 122. | Nussbaumer-Ochsner Y, Rabe KF. Systemic manifestations of COPD. Chest 2011;139:165–173. |
| 123. | Doyle TJ, Washko GR, Fernandez IE, Nishino M, Okajima Y, Yamashiro T, Divo MJ, Celli BR, Sciurba FC, Silverman EK, et al. Interstitial lung abnormalities and reduced exercise capacity. Am J Respir Crit Care Med 2012;185:756–762. |
| 124. | Pinto-Plata V, Casanova C, Müllerova H, de Torres JP, Corado H, Varo N, Cordoba E, Zeineldine S, Paz H, Baz R, et al. Inflammatory and repair serum biomarker pattern: association to clinical outcomes in COPD. Respir Res 2012;13:71. |
| 125. | Cockayne DA, Cheng DT, Waschki B, Sridhar S, Ravindran P, Hilton H, Kourteva G, Bitter H, Pillai SG, Visvanathan S, et al. Systemic biomarkers of neutrophilic inflammation, tissue injury and repair in COPD patients with differing levels of disease severity. PLoS ONE 2012;7:e38629. |
| 126. | Oudijk E-JD, Nijhuis EH, Zwank MD, van de Graaf EA, Mager HJ, Coffer PJ, Lammers JW, Koenderman L. Systemic inflammation in COPD visualised by gene profiling in peripheral blood neutrophils. Thorax 2005;60:538–544. |
| 127. | Yoshida K, Kondo R, Wang Q, Doerschuk CM. Neutrophil cytoskeletal rearrangements during capillary sequestration in bacterial pneumonia in rats. Am J Respir Crit Care Med 2006;174:689–698. |
| 128. | Summers C, White J, Singh N, Mackenzie I, Johnston A, Solanki C, Balan K, Peters AM, Chilvers ER. Establishing the pulmonary transit time of primed and unprimed neutrophils in man. Thorax 2009;64:A3. |
| 129. | Juss JK, Hayhoe RP, Owen CE, Bruce I, Walmsley SR, Cowburn AS, Kulkarni S, Boyle KB, Stephens L, Hawkins PT, et al. Functional redundancy of class i phosphoinositide 3-kinase (PI3K) Isoforms in signaling growth factor-mediated human neutrophil survival. PLoS ONE 2012;7:e45933. |
| 130. | Rytilä P, Rehn T, Ilumets H, Rouhos A, Sovijärvi A, Myllärniemi M, Kinnula VL. Increased oxidative stress in asymptomatic current chronic smokers and GOLD stage 0 COPD. Respir Res 2006;7:69. |
| 131. | Makris D, Vrekoussis T, Izoldi M, Alexandra K, Katerina D, Dimitris T, Michalis A, Tzortzaki E, Siafakas NM, Tzanakis N. Increased apoptosis of neutrophils in induced sputum of COPD patients. Respir Med 2009;103:1130–1135. |
| 132. | Brown V, Elborn JS, Bradley J, Ennis M. Dysregulated apoptosis and NFkappaB expression in COPD subjects. Respir Res 2009;10:24. |
| 133. | Menon MK, Houchen L, Singh SJ, Morgan MD, Bradding P, Steiner MC. Inflammatory and satellite cells in the quadriceps of patients with COPD and the response to resistance training. Chest 2012;142:1134–1142. |
Supported by the British Lung Foundation, the Medical Research Council, and the Cambridge NIHR-BRC.
Originally Published in Press as DOI: 10.1165/rcmb.2012-0492TR on January 17, 2013
Author disclosures are available with the text of this article at www.atsjournals.org.
