help button home button
AJRCCM
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Published ahead of print on November 16, 2006, doi:10.1164/rccm.200507-1152OC
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Online Supplement
Right arrow All Versions of this Article:
200507-1152OCv1
175/5/473    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yoshikawa, T.
Right arrow Articles by Djukanovic, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yoshikawa, T.
Right arrow Articles by Djukanovic, R.
American Journal of Respiratory and Critical Care Medicine Vol 175. pp. 473-479, (2007)
© 2007 American Thoracic Society
doi: 10.1164/rccm.200507-1152OC

Original Article

Impaired Neutrophil Chemotaxis in Chronic Obstructive Pulmonary Disease

Takahiro Yoshikawa1,2, Gordon Dent1,3, Jon Ward1, Gilbert Angco1, Guangmin Nong1, Naho Nomura2, Kazuto Hirata2 and Ratko Djukanovic1

1 Allergy and Inflammation Research, Division of Infection, Inflammation and Repair, University of Southampton School of Medicine, Southampton, United Kingdom; 2 Department of Respiratory Medicine, Graduate School of Medicine, Osaka City University, Osaka, Japan; and 3 Human Disease and Genomics, Institute of Science and Technology in Medicine, Keele University, Keele, United Kingdom

Correspondence and requests for reprints should be addressed to Ratko Djukanovic, D.M., F.R.C.P., Allergy & Inflammation Research, Division of Infection, Inflammation, and Repair, Mailpoint 810, Level F, South Block, University of Southampton School of Medicine, Southampton General Hospital, Southampton SO16 6YD, UK. E-mail: r.djukanovic{at}soton.ac.uk


   ABSTRACT
TOP
 ABSTRACT
AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rationale: Neutrophilic airway inflammation is considered to be a major factor in the pathogenesis of chronic obstructive pulmonary disease (COPD), with sputum and bronchoalveolar lavage neutrophil counts broadly correlating with disease severity. The mechanisms responsible for neutrophil accumulation are poorly understood, but they could involve increased influx and/or survival of these cells.

Objectives: To investigate whether neutrophil chemotactic responsiveness and/or chemotactic activity in airway secretions are increased in subjects with COPD.

Methods: Chemotaxis experiments were performed using induced sputum supernatants from subjects with and without COPD as a source of chemotactic activity, and neutrophils from healthy donors as responder cells. In addition, chemotactic responses to N-formyl-Met-Leu-Phe (fMLP) and interleukin-8 (IL-8/CXCL8) were studied using neutrophils from healthy subjects and subjects with COPD.

Measurements and Main Results: As reported in the literature, sputum neutrophil counts were significantly increased in subjects with COPD compared with healthy subjects. However, this was associated with reduced chemotactic activity in sputum in COPD, as judged by reduced chemotaxis to the fluid phase of sputum from subjects with COPD compared with healthy subjects. Furthermore, whereas neutrophils from subjects with stage I COPD had normal responses to fMLP and IL-8, subjects with more severe stage II–IV COPD showed reduced levels of spontaneous migration and chemotaxis to fMLP and IL-8.

Conclusions: Neither increased chemotactic activity in the airways nor increased chemotactic responsiveness of neutrophils explains the increased number of these cells in subjects with stable COPD. The implications of the observed reduction in neutrophil chemotactic activity remain to be established.

Key Words: chronic obstructive pulmonary disease • sputum • neutrophils • chemotaxis



   AT A GLANCE COMMENTARY
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Scientific Knowledge on the Subject
COPD is associated with airway neutrophilia. The mechanisms by which neutrophils accumulate in the airways of patients with COPD are not known.

What This Study Adds to the Field
Neither neutrophil chemotactic responsiveness nor chemotactic activity of airway secretions is elevated in COPD.

 
Chronic obstructive pulmonary disease (COPD) is associated with persistent neutrophilic inflammation of the airways and lung parenchyma (1, 2). Studies using induced sputum (3), bronchoalveolar lavage (3, 4), and resected lung tissue (5) have consistently shown that neutrophils infiltrate the airways and alveoli and are activated to release such mediators as myeloperoxidase and neutrophil lipocalin (6). Neutrophilia of both the large (7) and small airways (5) correlates broadly with disease severity, but the underlying mechanisms whereby these cells accumulate and how these processes are related to disease severity are unknown. Elevated sputum levels of the neutrophil chemoattractants interleukin (IL)-8 (CXCL8), tumor necrosis factor (TNF)-{alpha} (8), and growth-related oncogene (GRO)-{alpha} (CXCL1) (9) have been reported in studies of stable COPD, providing one plausible mechanism but not a definitive causal link. Although there are reports of significant correlations between sputum levels of mediators (e.g., IL-8) and COPD severity, as judged by FEV1 (10), there have been no reports of direct correlations between neutrophil counts and concentrations of mediators known to induce chemotaxis of neutrophils.

Studies of COPD and other airway diseases such as cystic fibrosis and bronchiectasis have shown prominent neutrophil chemotactic activity in the fluid phase of sputum (11–14). Application of neutralizing antibodies against IL-8 or use of antagonists for leukotriene B4 (LTB4) has demonstrated the contribution of these mediators to the process of neutrophil accumulation (11–14). However, these studies did not establish relationships between chemotactic activities and either airway neutrophilia or disease severity, nor did they compare the activity in subjects with COPD with that in healthy individuals. Only one study demonstrated a chemotactic role for LTB4, which was elevated in sputum during an infective exacerbation of chronic bronchitis and reduced to normal levels after successful treatment (15).

The present study was designed to test two main hypotheses: (1) that airway neutrophilia in COPD is due to enhanced influx of neutrophils from blood into the airways in response to chemoattractants produced by inflammatory and structural cells of the airways and (2) that the chemotactic activity is related to the severity of COPD. It is well established that cigarette smoke is a major cause of COPD, but it is unclear why only 15% of smokers develop COPD (16). Some (8), but not all (17), studies in smokers with normal lung function have shown raised sputum neutrophil counts, and no long-term studies have been conducted to investigate whether these subjects are at increased risk of developing COPD. We have, therefore, also hypothesized that smokers with and without airway obstruction can be differentiated by increased chemotactic activity in COPD and that this leads to an increased influx of neutrophils. By classifying the smokers according to the criteria set by the Global Initiative for Chronic Obstructive Lung Disease (GOLD) (18), we have sought to understand whether there is any relationship between the chemotactic activity and disease severity.


   METHODS
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Study Design
Volunteers were categorized into healthy subjects and subjects with COPD on the basis of clinical history and lung function tests. Peripheral blood neutrophils from each volunteer were assessed for chemotactic responsiveness to standard chemoattractants; sputum samples from volunteers were also assayed for neutrophil chemotactic activity.

Subjects
Recruited volunteers were classified as healthy nonsmokers (n = 17), healthy smokers (n = 16), patients with COPD stage I (n = 9), or patients with COPD stage II–IV (n = 19) according to established guidelines and on the basis of detailed history and lung function testing (18, 19). Details of the classification criteria are given in the online supplement. All subjects provided blood neutrophils for chemotaxis experiments (Table 1). A subgroup of these subjects (9 healthy nonsmokers, 10 healthy smokers, 5 subjects with stage I COPD, and 9 subjects with stage II–IV COPD) also provided sputum samples that met our quality criterion of containing fewer than 30% squamous cells (Table 2).


View this table:
[in this window]
[in a new window]

  		TABLE 1. CHARACTERISTICS OF SUBJECTS DONATING BLOOD NEUTROPHILS

 

View this table:
[in this window]
[in a new window]

  		TABLE 2. DIFFERENTIAL SPUTUM CELL COUNTS IN SUBJECTS WHO DONATED SPUTUM

 
The study was approved by the Southampton and South Wessex Local Research Ethics Committee, and all volunteers gave written, informed consent.

Sputum Induction and Processing
Sputum was induced and processed as recommended by the European Respiratory Society's Task Force on Induced Sputum (20). Although all subjects had a history of chronic bronchitis, none of the samples were obtained spontaneously (i.e., without the use of saline). Mucoid portions of sputum samples were selected to reduce salivary contamination and processed as described (21). Details of processing are provided in the online supplement.

Isolation and Fluorescent Labeling of Peripheral Blood Neutrophils
Neutrophils were isolated from blood using dextran sedimentation and separation on Lymphoprep (Axis-Shield UK, Kimbolton, Cambridgeshire, UK), followed by hypotonic lysis of residual red blood cells (22) (see the online supplement). In separate experiments we showed that this method of purification yielded neutrophils with chemotactic responses to a wide range of IL-8 and N-formyl-Met-Leu-Phe (fMLP) concentrations that were almost identical to responses observed when centrifugation on Percoll (GE Healthcare, Little Chalfont, Buckinghamshire, UK), often used in neutrophil studies (23), was applied for neutrophil purification. Details of this comparison are provided in the online supplement (Figure E4). Neutrophils (> 95% pure, > 98% viable, as determined by trypan blue exclusion) were labeled with the fluorescent vital dye calcein according to the method described by Taylor and colleagues (24). Details are provided in the online supplement.

Neutrophil Chemotaxis
Chemotaxis of calcein-labeled neutrophils was measured in a 96-well microplate blind-chamber system as described (24). Numbers of cells migrating through 3-µm-pore–size filter membranes were quantified using an intraexperiment standard curve for known numbers of labeled neutrophils. Further details are provided in the online supplement.

Spontaneous migration and chemotactic responses to a range of concentrations of fMLP and IL-8 were quantified for neutrophils obtained from each volunteer, and responses to the optimal concentrations were compared among volunteer groups. In separate experiments, the reproducibility of blood neutrophil responses to the full range of concentrations of fMLP and IL-8 was tested and found to be very consistent on three separate occasions when using neutrophils from two donors (see Figure E3).

Sputum induction was performed in a subset of volunteers (Table 2). The chemotactic activity of the generated sputum supernatants was first quantified using neutrophils from a single donor (experiment series 1) to avoid variability in responses due to responder cell differences. Chemotaxis experiments were then repeated using the same sputum samples but this time taking cells from seven nonsmoking healthy donors (experiment series 2) to make sure that the responses of neutrophils from the single donor were not unique to that donor. Details are given in the online supplement.

Neutrophil migration is expressed as total numbers of cells migrating in 1 hour. Chemotactic responses to fMLP, IL-8, and sputum supernatants are expressed as net numbers of migrating cells with spontaneous migration subtracted.

Statistical Analysis
All statistical analyses were performed using InStat for Windows, version 3.06 (GraphPad Software, Inc., San Diego, CA). Subjects' clinical data are presented as mean (SD). Sputum cell numbers and neutrophil migration data, which were not normally distributed, are presented as median (interquartile range). Chemotaxis data for the four volunteer groups were first compared by one-way analysis of variance; where this returned a significant difference among groups, pairwise comparisons were performed using the Student-Newman-Keuls test. Sputum cell counts for the four groups, which were not normally distributed, were compared by Kruskal-Wallis test, with post hoc pairwise comparisons, where appropriate, performed by Dunn's test. The reproducibility of the sputum-induced chemotactic responses, which used single and multiple neutrophil donors, was assessed by performing Bland-Altman analysis on the data from series 1 and 2 experiments. Correlation between lung function parameters and neutrophil counts/chemotactic activity was performed by Spearman rank correlation. In each case, a p value of less than 0.05 was defined as significant.


   RESULTS
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Induced Sputum Cell Counts
Subjects with COPD stage II–IV had higher sputum neutrophil counts than the healthy nonsmokers or healthy smokers, and sputum neutrophil counts were also significantly higher in subjects with COPD stage I than in healthy nonsmokers (Table 2). There was a significant negative correlation between neutrophil counts (expressed as % of total inflammatory cells) and FEV1 (% predicted; rS = –0.859, p < 0.0001; see Figure E1).

Chemotaxis of Blood Neutrophils Induced by fMLP and IL-8
In view of previous reports of altered chemotactic responses of circulating neutrophils in COPD (25), peripheral blood neutrophils from the four groups of volunteers were first analyzed for their ability to migrate in response to two established chemotactic factors: fMLP and IL-8. The magnitude of neutrophil chemotaxis was maximal at the concentration of 10–8 M for fMLP and 10–9 to 10–8 M for IL-8 in all experiments (Figure 1), and responsiveness to both stimuli showed a similar overall reduction in the COPD stage II–IV group. The optimal concentration of 10–8 M of each stimulus was then used for comparison of the subject groups. The numbers of cells migrating toward fMLP (corrected for spontaneous migration) were lower in subjects with COPD stage II–IV than those from healthy nonsmoking control subjects (p < 0.001), healthy smokers (p < 0.001), or subjects with COPD stage I (p < 0.01; Figure 2). Similarly, chemotactic responses to IL-8 were also reduced in the stage II–IV COPD group when compared with COPD stage I (p < 0.05) or healthy nonsmokers (p < 0.01; Figure 2). There was a significant correlation between FEV1 (% predicted) and the chemotactic response to fMLP, but not IL-8 (rS = 0.524, p < 0.001; see Figure E2).


Figure 1
View larger version (15K):
[in this window]
[in a new window]

  		Figure 1. Neutrophil migration induced by N-formyl-Met-Leu-Phe (fMLP) (top panel) and IL-8 (bottom panel). Numbers of cells migrating to fMLP or IL-8 are shown as median values for each group after subtracting the numbers of spontaneously migrating cells: healthy nonsmokers (open circles), healthy smokers (open triangles), subjects with chronic obstructive pulmonary disease (COPD) stage I (closed circles), and subjects with COPD stage II–IV (closed triangles).

 

Figure 2
View larger version (17K):
[in this window]
[in a new window]

  		Figure 2. Neutrophil migration in response to optimal concentrations (10–8 M) of (A) fMLP and (B) IL-8. Chemotactic responses are shown as individual data points. Horizontal bars represent mean values.

 
Chemotaxis of Blood Neutrophils Induced by the Sputum Fluid Phase
All sputum samples exhibited significant chemotactic activity for neutrophils. Dilution-response curves were not uniformly bell-shaped, but showed a dilution-dependent response (see Figure E5). In both series 1 (using single blood donor) and 2 (using several donors) experiments, the neutrophil chemotactic response to sputum supernatants (diluted 1:10) from subjects with COPD stage II–IV or stage I was reduced when compared with sputum from nonsmoking control subjects (Figure 3). There was good reproducibility between the series 1 and 2 experiments regarding the chemotactic activity in sputum (Figure 4 and Figure E6).


Figure 3
View larger version (9K):
[in this window]
[in a new window]

  		Figure 3. Neutrophil migration in response to sputum (diluted 1:10) of individual donors of sputum. (A) In the first experiment series, a single healthy nonsmoker was used as the donor of neutrophils for the chemotaxis experiments. (B) In the second series, seven neutrophil donors were used. Horizontal bars represent median values. Bland-Altman analysis of series 1 and series 2 experiments showed high reproducibility (see the online supplement).

 

Figure 4
View larger version (26K):
[in this window]
[in a new window]

  		Figure 4. The reproducibility between the series 1 and 2 experiments regarding the chemotactic activity in sputum (diluted 1:40, 1:20, 1:10) for each series of experiments. Light-gray shading, the first series of experiments; dark-gray shading, the second series of experiments.

 
There was a significant positive correlation between the chemotactic activity of sputum fluid phase and FEV1 (rS = 0.544, p = 0.002; see Figure E7).


   DISCUSSION
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study was designed to assess the migratory capacity of circulating neutrophils in smokers with and without COPD and the chemotactic properties of airway secretions from the same subjects who displayed varying degrees of clinical severity and airway neutrophilia. In agreement with previous reports (6, 9, 17), sputum samples from subjects with moderate to severe COPD contained more neutrophils than samples from healthy nonsmokers and smokers. However, contrary to our main hypothesis, blood neutrophils from subjects with moderate to severe COPD showed decreased chemotaxis in response to fMLP and IL-8 when compared with blood neutrophils from healthy nonsmokers and subjects with mild COPD. The fMLP-induced migration correlated positively with FEV1, further suggesting that this was a phenomenon that was associated with disease severity: reduced lung function was associated with lower levels of neutrophil chemotactic responsiveness. In further contradiction of our hypothesis, the chemotactic activity present in the sputum fluid phase from subjects with moderate to severe COPD was reduced when compared with sputum from both healthy nonsmokers and healthy smokers whether using neutrophils from a single donor or multiple donors as responder cells in the chemotaxis experiments.

The finding of reduced neutrophil chemotaxis of blood neutrophils in the present study is surprising given previous reports of increased expression of fMLP receptors on neutrophils from subjects with emphysema (26). It is also unexpected in view of reports, albeit in an animal model, that nicotine enhances chemotactic responses to fMLP (27), and the fact that all the subjects with COPD in the current study were smokers. The results of our study are also in disagreement with those from Burnett and colleagues (25) who found that neutrophils from subjects with chronic bronchitis and emphysema have increased chemotactic responses to fMLP. Although we cannot exclude the possibility that some of this is due to different subjects studied, the discrepancy between the current study and that by Burnett and colleagues could be due to different methods used to quantify chemotaxis. In the current study, cell migration was assessed using an observer-independent method, which is likely to be less observer biased than methods that depend on visual counting.

We do not think that the method of quantification itself explains the discrepancy. Our group (28) and others (29) have reported that labeling of neutrophils with calcein does not affect their migration. Furthermore, activation of these cells with IL-8, phorbol 12-myristate 13-acetate (PMA), and C5a does not affect the fluorescence due to calcein labeling (29). Finally, the overall responsiveness of neutrophils in the present study is consistent with previous reports, with maximal responses of neutrophil chemotaxis for both IL-8 and fMLP at 10–8 M (30, 31). Our finding of reduced neutrophil function is in accordance with the finding by Fietta and coworkers (32) of a reduction in a number of functions of monocytes and neutrophils in COPD, including chemotaxis, and phagocytosis and killing of Candida albicans.

Having found a reduction in chemotactic responses of blood-derived neutrophils that was related to disease severity, we proceeded to study whether there are any differences in chemotactic activity in sputum that might explain the increased numbers of neutrophils in sputum in smokers with COPD. To control for any differences in responsiveness of neutrophils obtained from individuals with varying disease, in the first series of experiments we chose to use cells from a single healthy donor who had been found previously to have consistent responses to fMLP and IL-8. These experiments were complemented with a second series of experiments in which neutrophils from several healthy donors were used as responder cells but we used the same sputum samples as in the first series as a source of chemotactic activity. All the sputum samples had chemotactic activity for neutrophils, resulting in a dilution-dependent response that was in keeping with responses previously reported when using sputum (12, 14, 15). As with blood neutrophil responsiveness, and contrary to our hypothesis, the chemotactic activity in sputum was reduced in COPD. Theoretically, this could be explained either by a reduction in chemoattractants or by the presence of endogenous inhibitors, such as chemotactic factor inactivator (33), or, in the case of IL-8, secretory component (34). Elucidation of the mechanisms that could be involved would require looking beyond the known chemoattractants for neutrophils. Airway secretions obtained from individuals with COPD contain neutrophil chemotactic factors—such as IL-8 and LTB4—which, based on published data, only account for approximately 30% of chemotactic activity (13). The assessment of the contribution of individual mediators and the identification of factors that could inhibit neutrophil migration were beyond the scope of this study.

This study adds to the growing evidence that progression of COPD is not necessarily associated with more inflammatory cell activity, at least in the large airways. Our findings are in keeping with observations made by others of reduced inflammatory cell activity in this disease. Recently, Hodge and colleagues have found that alveolar macrophages from subjects with COPD exhibit impaired phagocytosis of apoptotic epithelial cells, which could account for the increased numbers of such cells in the lungs in COPD (35, 36). The same authors have also shown increased numbers of apoptotic T lymphocytes in COPD (36). Another study comparing subjects with COPD and healthy smokers has shown reduced numbers of {gamma}{delta} T lymphocytes (37), cells that play a role in tissue repair and mucosal homeostasis (38). This suggested that smokers who go on to develop COPD fail to mount a regulatory response involving {gamma}{delta} T lymphocytes. The same group found that, when compared with smokers with normal lung function and nonsmokers, surface expression of HLA-DR and CD80 was lower on alveolar macrophages from subjects with COPD and that these subjects also had a higher percentage of macrophages with a low-level surface expression of CD44 (39). These receptors reflect the functional capacity of macrophages to phagocytose and present antigen.

This study suggests that neutrophil accumulation in COPD airways cannot be explained by increased neutrophil chemotactic activity in the airway secretions, although we cannot exclude the possibility of increased activity in the tissue. Other factors that need to be considered are a change in chemoattractant receptor expression on neutrophils residing in the mucosa, something that does not lend itself readily to analysis, and interaction between the neutrophils and endothelial cells and/or epithelial cells. To our knowledge, there have been no studies to date looking at possible interactions between neutrophils from subjects with COPD and epithelial cells, although reduced expression of secretory leukoprotease inhibitor (SLPI) at sites of squamous metaplasia in the airways of patients with COPD may lead to heightened protease-dependent interactions (40). A recent study by Woolhouse and colleagues (41) has shown that neutrophils from subjects with COPD interact to a greater extent than "healthy" neutrophils with human umbilical vein endothelial cells, and it is possible that a similar interaction in the airways in COPD might enhance recruitment of cells via increased endothelial adhesion and transendothelial migration. We also cannot exclude differences in activity between proximal and more distal airways, where prominent neutrophilia is seen (5). Neutrophils could also accumulate in the airways as a result of prolonged survival. Apoptosis is the major mechanism controlling the longevity of neutrophils at inflamed sites (42); the chronic inflammatory microenvironment (or smoking itself) might inhibit the normal recognition mechanisms whereby monocyte-derived macrophages can engulf apoptotic neutrophils (43, 44), although there is, as yet, no definitive mechanism for these effects.

Some of our interpretations may be limited by the fact that we did not assess bacterial colonization of the airways to the extent done previously by other authors who showed the presence of colonizing bacteria in the airways of patients with clinically stable COPD (45–47). Bacteriologic screening of volunteers' sputum samples was conducted using standard protocols and criteria for clinical relevance applied in the public health laboratory of the hospital where the study was performed. This was performed at the time of recruitment to the study and showed no significant growth of pathogenic bacteria, excluding significant, clinically relevant infection. However, more detailed quantitative methods for the assessment of colonization, reported by other authors to show colonization in COPD (48, 49), were not applied. Furthermore, blood and sputum sampling for the chemotaxis experiments took place at varying times after the initial screening. Therefore, we cannot exclude the possibility that differences in current bacterial colonization of the airways at the time of sampling might have influenced the chemotactic responses of the blood-derived neutrophils because exposure to bacterial chemoattractants may possibly suppress neutrophil responsiveness to chemokines (50). However, this explanation would be in contradiction to studies that have shown that bacterial numbers in stable COPD are associated with increased markers of airway inflammation (49, 51, 52), including the neutrophil-active chemokine IL-8. Furthermore, whereas bacterial load may influence negatively the chemotactic responses of circulating neutrophils from subjects with COPD, it is difficult for the same argument to explain the reduced chemotactic activity observed in the sputum of subjects with COPD because the neutrophils used in the sputum chemotaxis experiments were derived from healthy individuals.

In summary, we have found evidence of reduced neutrophil chemotactic activity in subjects with moderate to severe COPD and conclude, therefore, that mechanisms other than chemotaxis contribute to the increased airway neutrophilia that is typical of COPD and is related to reduced lung function. Our findings add to the accumulating evidence that more disease does not necessarily mean more cellular activity. Further studies are needed to elucidate the mechanisms whereby increased numbers of neutrophils accumulate in the airways of subjects with COPD and to identify factors that are responsible for the observed reduction in neutrophil chemotaxis.


   FOOTNOTES
 
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Originally Published in Press as DOI: 10.1164/rccm.200507-1152OC on November 16, 2006

Conflict of Interest Statement: T.Y. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. G.D. received $180,000 in 2002 from Bayer, Inc., as a sponsorship for basic research. J.W. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. G.A. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. G.N. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. N.N. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. K.H. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. R.D. has received sponsorship from AstraZeneca for basic research (£686,000) and from GlaxoSmithKline for participation in a multicenter clinical trial (£348,000); he is a cofounder of Synairgen PLC, holding stock worth £60,000 and receiving £15,000 per annum in consultancy fees.

Received in original form July 26, 2005; accepted in final form November 16, 2006


   REFERENCES
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Saetta M. Airway inflammation in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1999;160(Suppl):S17–S20.[Abstract/Free Full Text]
  2. Barnes PJ. Medical progress: chronic obstructive pulmonary disease. N Engl J Med 2000;343:269–280.[Free Full Text]
  3. Rutgers SR, Timens W, Kaufmann HF, van der Mark TW, Koeter GH, Postma DS. Comparison of induced sputum with bronchial wash, bronchoalveolar lavage and bronchial biopsies in COPD. Eur Respir J 2000;15:109–115.[Abstract]
  4. 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 J Respir Crit Care Med 1985;132:254–260.
  5. Hogg JC, Chu F, Utokaparch S, Woods R, Elliott WM, Buzatu L, Cherniak RM, Rogers RM, Sciurba FC, Coxson HO, et al. The nature of small-airway obstruction in chronic obstructive pulmonary disease. N Engl J Med 2005;350:2645–2653.
  6. 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.[Abstract]
  7. 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.[Abstract/Free Full Text]
  8. Keatings VM, Collins PD, Scott DM, Barnes PJ. Differences in interleukin-8 and tumor necrosis factor-{alpha} in induced sputum from patients with chronic obstructive pulmonary disease or asthma. Am J Respir Crit Care Med 1996;153:530–534.[Abstract]
  9. Traves SL, Culpitt SV, Russell RE, Barnes PJ, Donnelly LE. Increased levels of the chemokines GRO{alpha} and MCP-1 in sputum samples from patients with COPD. Thorax 2002;57:590–595.[Abstract/Free Full Text]
  10. Yamamoto C, Yoneda T, Yoshikawa M, Fu A, Tokuyama T, Tsukaguchi K, Narita N. Airway inflammation in COPD assessed by sputum levels of interleukin-8. Chest 1997;112:505–510.
  11. Richman-Eisenstat JBY, Jorens PG, Hebert CA, Ueki I, Nadel JA. Interleukin-8: an important chemoattractant in sputum of patients with chronic inflammatory airway diseases. Am J Physiol 1993;264:L413–L418.
  12. Mikami M, Llewellyn-Jones CG, Bayley D, Hill SL, Stockley RA. The chemotactic activity of sputum from patients with bronchiectasis. Am J Respir Crit Care Med 1998;157:723–728.[Abstract/Free Full Text]
  13. Woolhouse IS, Bayley DL, Stockley RA. Sputum chemotactic activity in chronic obstructive pulmonary disease: effect of {alpha}1-antitrypsin deficiency and the role of leukotriene B4 and interleukin 8. Thorax 2002;57:709–714.[Abstract/Free Full Text]
  14. Beeh KM, Kornmann O, Buhl R, Culpitt SV, Giembycz MA, Barnes PJ. Neutrophil chemotactic activity of sputum from patients with COPD: role of interleukin 8 and leukotriene B4. Chest 2003;123:1240–1247.
  15. Crooks SW, Bayley DL, Hill SL, Stockley RA. Bronchial inflammation in acute bacterial exacerbations of chronic bronchitis: the role of leukotriene B4. Eur Respir J 2000;15:274–280.[Abstract]
  16. Fletcher C, Peto R. The natural history of chronic airflow obstruction. BMJ 1977;1:1645–1648.[Medline]
  17. O'Donnell RA, Richter A, Ward J, Angco G, Mehta A, Rousseau K, Swallow DM, Holgate ST, Djukanovic R, Davies DE, et al. Expression of ErbB receptors and mucins in the airways of long term current smokers. Thorax 2004;59:1032–1040.[Abstract/Free Full Text]
  18. Pauwels RA, Buist AS, Calverley PMA, Jenkins CR, Hurd SS. Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease: NHLBI/WHO Global Initiative for Chronic Obstructive Lung Disease (GOLD) workshop summary. Am J Respir Crit Care Med 2001;163:1256–1276.[Free Full Text]
  19. National Heart, Lung, and Blood Institute. Global Initiative for Chronic Obstructive Lung Disease: global strategy for diagnosis, management, and prevention of chronic obstructive pulmonary disease (2004 update). Bethesda, MD: National Institutes of Health; 2004.
  20. Paggiaro PL, Chanez P, Holz O, Ind PW, Djukanovic R, Maestrelli P, Sterk PJ. Sputum induction. Eur Respir J 2002;3s–8s.
  21. Hadjicharalambous C, Dent G, May RD, Handy RLC, Anderson IK, Davies DE, Djukanovic R. Measurement of eotaxin (CCL11) in induced sputum supernatants: validation and detection in asthma. J Allergy Clin Immunol 2004;113:657–662.[CrossRef][Medline]
  22. Böyum A. Isolation of mononuclear cells and granulocytes from human blood: isolation of mononuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g. Scand J Clin Lab Invest 1968;77–89.
  23. Haslett C, Guthrie LA, Kopaniak MM, Johnson RB Jr, Henson PM. Modulation of multiple neutrophil functions by preparative methods or trace concentrations of bacterial lipopolysaccharide. Am J Pathol 1985;119:101–110.[Abstract]
  24. Taylor ML, Dastych J, Sehgal D, Sundstrom M, Nilsson G, Akin C, Mage RG, Metcalfe DD. The Kit-activating mutation D816V enhances stem cell factor-dependent chemotaxis. Blood 2001;98:1195–1199.[Abstract/Free Full Text]
  25. Burnett D, Chamba A, Hill SL, Stockley RA. Neutrophils from subjects with chronic obstructive lung disease show enhanced chemotaxis and extracellular proteolysis. Lancet 1987;2:1043–1046.[Medline]
  26. Stockley RA, Grant RA, Llewellyn-Jones CG, Hill SL, Burnett D. Neutrophil formyl-peptide receptors: relationship to peptide-induced responses and emphysema. Am J Respir Crit Care Med 1994;149:464–468.[Abstract]
  27. Nowak D, Ruta U, Piasecka G. Nicotine increases human polymorphonuclear leukocytes chemotactic response: a possible additional mechanism of lung injury in cigarette smokers. Exp Pathol 1990;39:37–43.[Medline]
  28. Dent G, Hadjicharalambous C, Yoshikawa T, Handy RLC, Powell J, Anderson IK, Louis R, Davies DE, Djukanovic R. Contribution of eotaxin-1 to eosinophil chemotactic activity of moderate and severe asthmatic sputum. Am J Respir Crit Care Med 2004;169:1110–1117.[Abstract/Free Full Text]
  29. Frevert CW, Wong VA, Goodman RB, Goodwin R, Martin TR. Rapid, fluorescence-based measurement of neutrophil migration in vitro. J Immunol Methods 1998;213:41–52.[CrossRef][Medline]
  30. Tanaka S, Robinson EA, Yoshimura T, Matsushima K, Leonard EJ, Appella E. Synthesis and biological characterization of monocyte-derived neutrophil chemotactic factor. FEBS Lett 1998;236:467–470.
  31. Showell HJ, Freer RJ, Zigmond SH, Schiffman E, Aswaikumar S, Corcoran B, Becker EL. The structure-activity relations of synthetic peptides as chemotactic factors and inducers of lysosomal secretion for neutrophils. J Exp Med 1976;143:1154–1169.[Abstract/Free Full Text]
  32. 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.[Medline]
  33. Robbins RA, Gossman GL, Nelson KJ, Koyama S, Thompson AB, Rennard SI. Inactivation of chemotactic factor inactivator by cigarette smoke: a potential mechanism of modulating neutrophil recruitment to the lung. Am Rev Respir Dis 1990;142:763–768.[Medline]
  34. Marshall LJ, Perks B, Ferkol T, Shute JK. IL-8 released constitutively by primary bronchial epithelial cells in culture forms an inactive complex with secretory component. J Immunol 2001;167:2816–2823.[Abstract/Free Full Text]
  35. 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.[CrossRef][Medline]
  36. Hodge S, Hodge G, Holmes M, Reynolds PN. Increased airway epithelial and T-cell apoptosis in COPD remains despite smoking cessation. Eur Respir J 2005;25:447–454.[Abstract/Free Full Text]
  37. Pons J, Sauleda J, Ferrer JM, Barcelo B, Fuster A, Regueiro V, Julia MR, Agusti AG. Blunted {gamma}{delta} T-lymphocyte response in chronic obstructive pulmonary disease. Eur Respir J 2005;25:441–446.[Abstract/Free Full Text]
  38. Jameson J. T-cell effector mechanisms: {gamma}{delta} and CD1d-restricted subsets. Curr Opin Immunol 2003;15:349–353.[CrossRef][Medline]
  39. Pons AR, Noguera A, Blanquer D, Sauleda J, Pons J, Agusti AG. Phenotypic characterisation of alveolar macrophages and peripheral blood monocytes in COPD. Eur Respir J 2005;25:647–652.[Abstract/Free Full Text]
  40. Aarbio J, van Schadewijk A, Stolk J, Sont JK, de Boer WI, Rabe KF, van Krieken JHJM, Mauad T, Hiemstra PS. Human neutrophil defensins and secretory leukocyte proteinase inhibitor in squamous metaplastic epithelium of bronchial airways. Inflamm Res 2004;53:230–238.[CrossRef][Medline]
  41. Woolhouse IS, Bayley DL, Lalor P, Adams DH, Stockley RA. Endothelial interactions of neutrophils under flow in chronic obstructive pulmonary disease. Eur Respir J 2005;25:612–617.[Abstract/Free Full Text]
  42. Haslett C. Granulocyte apoptosis and its roles in the resolution and control of lung inflammation. Am J Respir Crit Care Med 1999;160:S5–S11.[Abstract/Free Full Text]
  43. Whyte MKB, Meagher LC, Hardwick SJ, Savill JS, Haslett C. Transient elevations of cytosolic free calcium retard subsequent apoptosis in neutrophils in vitro. J Clin Invest 1993;92:446–455.[Medline]
  44. Rossi AG, Cousin JM, Dransfield I, Lawson MF, Chilvers ER, Haslett C. Agents that elevate cAMP inhibit human neutrophil apoptosis. Biochem Biophys Res Commun 1995;217:892–899.[CrossRef][Medline]
  45. Monso E, Ruiz J, Rosell A, Manterola J, Fiz J, Morera J, Ausina V. Bacterial infection in chronic obstructive pulmonary disease: a study of stable and exacerbated outpatients using the protected specimen brush. Am J Respir Crit Care Med 1995;152:1316–1320.[Abstract]
  46. Monso E, Rosell A, Bonet G, Manterola J, Cardona PJ, Ruiz J, Morera J. Risk factors for lower airway bacterial colonization in chronic bronchitis. Eur Respir J 1999;13:338–342.[Abstract]
  47. Zalacain R, Sobradillo V, Amilibia J, Barron J, Achotegui V, Pijoan JI, Llorente JL. Predisposing factors to bacterial colonization in chronic obstructive pulmonary disease. Eur Respir J 1999;13:343–348.[Abstract]
  48. 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.[Abstract/Free Full Text]
  49. Hill AT, Campbell EJ, Sill 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.[CrossRef][Medline]
  50. Heit B, Tavener S, Raharjo E, Kubes P. An intracellular signalling hierarchy determines direction of migration in opposing chemotactic gradients. J Cell Biol 2002;159:91–102.[Abstract/Free Full Text]
  51. Sethi S, Murphy TF. Bacterial infection in chronic obstructive pulmonary disease in 2000: a state-of-the-art review. Clin Microbiol Rev 2001;14:336–363.[Abstract/Free Full Text]
  52. Wilkinson TM, Patel IS, Wilks M, Donaldson GC, Wedzicha JA. Airway bacterial load and FEV1 decline in patients with chronic obstructive pulmonary disease. Am J Respir Respir Crit Care Med 2003;167:1090–1095.



This article has been cited by other articles:


Home page
 Eur Respir JHome page
K. F. Chung and I. M. Adcock
Multifaceted mechanisms in COPD: inflammation, immunity, and tissue repair and destruction
Eur. Respir. J., June 1, 2008; 31(6): 1334 - 1356.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
W. MacNee
Update in Chronic Obstructive Pulmonary Disease 2007
Am. J. Respir. Crit. Care Med., April 15, 2008; 177(8): 820 - 829.
[Full Text] [PDF]

Home page
 ChestHome page
T. Yamagata, H. Sugiura, T. Yokoyama, S. Yanagisawa, T. Ichikawa, K. Ueshima, K. Akamatsu, T. Hirano, M. Nakanishi, Y. Yamagata, et al.
Overexpression of CD-11b and CXCR1 on Circulating Neutrophils: Its Possible Role in COPD
Chest, September 1, 2007; 132(3): 890 - 899.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
J. Zhu, Y. Qiu, M. Valobra, S. Qiu, S. Majumdar, D. Matin, V. De Rose, and P. K. Jeffery
Plasma Cells and IL-4 in Chronic Bronchitis and Chronic Obstructive Pulmonary Disease
Am. J. Respir. Crit. Care Med., June 1, 2007; 175(11): 1125 - 1133.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Online Supplement
Right arrow All Versions of this Article:
200507-1152OCv1
175/5/473    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yoshikawa, T.
Right arrow Articles by Djukanovic, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yoshikawa, T.
Right arrow Articles by Djukanovic, R.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Proc. Am. Thorac. Soc. Am. J. Respir. Cell Mol. Biol.
Copyright © 2007 American Thoracic Society