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(Chest. 2004;125:1837-1842.)
© 2004 American College of Chest Physicians

Expression of Adhesion Molecules During Apoptosis of Circulating Neutrophils in COPD*

Aina Noguera, MD; Ernest Sala, MD; Antonia R. Pons, MD; Julio Iglesias, PhD; William MacNee, MD and Alvar G.N. Agustí, MD

* From the Serveis de Analisis Cliniques (Drs. Noguera and Pons), Pneumologia (Drs. Sala and Agustí), and Inmunologia (Dr. Iglesias), Hospital Universitari Son Dureta, Palma de Mallorca, Spain; and ELEGI Laboratories (Dr. MacNee), Royal Infirmary, Edinburgh, Scotland, UK.

Correspondence to: Aina Noguera, MD, Servei Analisis Cliniques, Hospital Universitari Son Dureta, Andrea Doria 55, 07014 Palma Mallorca, Spain; e-mail: anoguera{at}hsd.es


   Abstract
TOP
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
 References
 
Study objectives: Neutrophil accumulation occurs in the lungs of patients with COPD. This can be due to increased recruitment and/or delayed tissue clearance. Previous studies have described alterations in circulating neutrophils in these patients that can facilitate the former. Dysregulation of neutrophil apoptosis may contribute to the latter. This study investigated the potential abnormalities of the apoptotic process in COPD patients.

Design: Prospective study.

Settings: Outpatient clinic in a urban, tertiary hospital.

Patients: Fourteen stable patients with COPD, 8 smokers with normal lung function, and 8 healthy nonsmoking subjects.

Measurements and results: We cultured circulating neutrophils that had been harvested from the study subjects at 2, 6, and 24 h. Apoptosis was assessed using flow cytometry by annexin binding and CD16 expression. The surface expression of the adhesion molecules Mac-1 (CD11b) and L-selectin (CD62L) also was determined by flow cytometry. The percentage of apoptotic neutrophils increased with time similarly in all groups. However, the surface expression of Mac-1 (CD11b) was higher, and that of L-selectin (CD62L) was lower, during apoptosis in the neutrophils of patients with COPD.

Conclusions: These results show that, quantitatively, in vitro neutrophil apoptosis in COPD patients occurred at a rate similar to that found in healthy individuals and smokers with normal lung function. Qualitatively, however, the increased surface expression of Mac-1 (CD11b) and the decreased surface expression of L-selectin (CD62L) observed in the apoptotic neutrophils of COPD patients indicate increased activation during the apoptotic process. This may be relevant for the pathogenesis of COPD.

Key Words: chronic bronchitis • emphysema • inflammation • programmed cell death


   Introduction
TOP
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Conclusions
 References
 
COPD is characterized by an abnormal inflammatory response to respiratory pollutants, mostly from tobacco smoking.1 Neutrophil accumulation in the lungs is a prominent feature of this inflammatory response.2 Yet, the cellular and molecular mechanisms involved are poorly understood.

Neutrophil accumulation in tissues is a dynamic process that is governed, on the one hand, by the rate of neutrophil recruitment from the bloodstream and, on the other hand, by their rate of clearance.3 Previous studies from our laboratory45 and others67 have described abnormalities in the circulating neutrophils of patients with COPD that can facilitate their recruitment to the lungs, such as increased expression of the surface adhesion molecule CD11b (Mac-1). In contrast, to our knowledge, potential alterations in the mechanisms of neutrophil clearance have not been investigated in COPD patients.

Neutrophil clearance from tissues normally occurs by the engulfment and digestion of apoptotic neutrophils by macrophages.8 Under normal conditions, neutrophils are short-lived cells (with a half-life of < 1 day) that die as a result of the activation of the programmed cell death program.9 The latter, however, can be substantially modified by the local inflammatory milieu1011 and the activation state of the cell.12 Patients with COPD show increased levels of proinflammatory cytokines13 and upregulation of surface adhesion molecules.4 It is therefore possible that dysregulation of neutrophil apoptosis may occur in these patients, as it has been already reported in other inflammatory diseases.141516 So far, however, this has not been studied.

The assessment of neutrophil apoptosis in the lung is technically cumbersome. Yet, because of its potential pathogenic relevance, we decided to begin to address this issue by investigating potential abnormalities of neutrophil apoptosis in COPD patients in vitro. To this end, we first characterized the dynamics of neutrophil apoptosis in culture, in cells harvested from peripheral blood samples in patients with COPD, smokers with normal lung function, and never-smokers. Second, because the abnormal surface expression of Mac-1 (CD11b) and L-selectin (CD62L) reported in the circulating neutrophils of patients with COPD45 may influence neutrophil apoptosis,17 we also quantified by flow cytometry the expression of these two molecules during the apoptotic process.


   Materials and Methods
TOP
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Conclusions
 References
 
Population and Ethics
Patients with COPD (14 patients) were recruited from the outpatient clinic of our institution. All patients were considered to be clinically stable because none of them had required medical attention and/or a change in their regular therapy (ie, inhaled bronchodilators) during the previous 4 months. None of them had been treated with inhaled or oral corticosteroids. Patients with bronchial asthma, pneumonia, or lung cancer were specifically excluded. Smokers with normal lung function (eight subjects) were recruited from among individuals attending the pulmonary function laboratory of our hospital for a variety of clinical reasons, mostly preoperative assessment for noncancer surgery. All of them refrained from smoking for 12 h before blood sampling. Healthy never-smokers (eight subjects) were recruited by public advertisement. All participants gave their written consent after being fully informed of the nature, characteristics, risks, and potential benefits of the study, which had been previously approved by the ethics committee of our institution.

Neutrophil Isolation and Culture
Neutrophils were isolated from peripheral blood samples according to a method previously described in our laboratory.45 Briefly, after washing in phosphate-buffered saline solution, neutrophils were suspended at a concentration of 5 x 106 cells/mL in endotoxin-free Roswell Park Memorial Institute medium-1640/10% fetal calf serum, supplemented with 0.2 L glutamine, 25 U/mL penicillin, and 25 mg/mL streptomycin. Cells were separated in aliquots and cultured in sterile polypropylene tubes (Greiner; Frickenhausen, Germany) at 37°C and 5% CO2 for 2, 6, and 24 h. At these time points, we determined the rate of neutrophil apoptosis, and the level of surface expression of Mac-1 (CD11b) and L-selectin (CD62L).

Assessment of Neutrophil Apoptosis
The surface expression of phosphatidylserine and Fc{gamma}RIII (CD16) are standard markers of apoptosis.1819 Therefore, in our study we used flow cytometry to quantify the binding of annexin V (annexin V-fluorescein isothiocyanate; Bender MedSystems Diagnostics GmbH; Vienna, Austria) to phosphatidylserine, and the shedding of Fc{gamma}RIII (CD16) [clone 3G8; Immunotech; Marseille, France] on the neutrophil membrane as markers of apoptosis. In brief, at preestablished time points (2, 6, and 24 h of culture), neutrophils were washed once in binding buffer (10 mmol/L N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid/MaOH [pH 7.4], 140 mmol/L NaCl, and 2.5 mmol/L CaCl2), and 5 x 105 cells were incubated with 2.5 µL annexin V and 20 µL phycoerythrin-conjugated antihuman CD16 for 30 min at 4°C in the dark. Twenty-five thousand cells were analyzed in each sample. Cell viability was determined by flow cytometric analysis, which was performed after 2 min of incubation of the cell suspension (500 µL) with 10 µL propidium iodide solution (Bender MedSystems Diagnostics GmbH).

Assessment of Adhesion Molecule Expression
To assess the expression of Mac-1 (CD11b) and L-selectin (CD62L) on apoptotic neutrophils, we also used flow cytometry with double labeling with annexin V and phycoerythrin- conjugated CD11b (clone Bear 1; Immunotech) or CD62L (DREG56 clone; Immunotech). As negative controls, cells were stained with equivalent concentrations of irrelevant murine IgG1-Pe (Coulter Corporation; Miami, FL). In brief, after washing in binding buffer, cells were analyzed by FACScan flow cytometry (CellQuest; Becton Dickinson; Mountain View, CA) with standard settings.4 Twenty-five thousand cells were analyzed in each sample. Mean fluorescence intensity was measured as a marker of the cell surface expression of Mac-1 and L-selectin.

Statistical Analysis
A two-way analysis of variance for repeated measures (followed by post hoc contrasts [least significant differences], if appropriate) was used to assess the statistical significance of the differences observed between groups. A p value < 0.05 was considered to be significant.


   Results
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Conclusions
 References
 
Clinical Data
Table 1 shows the main clinical and lung function variables of all participants. All of them were men of similar age. The smoking history of patients with COPD was similar to that of smokers with normal lung function. None of the former was a current smoker (Table 1). COPD patients showed moderate-to-severe airflow obstruction and mild-to-moderate hypoxemia (Table 1). By design, the results of forced spirometry were normal in smokers with normal lung function and in healthy nonsmokers (Table 1).


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  		Table 1. Clinical and Lung Function Data of All Participants*

 
Dynamics of Apoptosis
Figure 1 shows the percentage of annexin V-positive neutrophils and the percentage of neutrophils with CD16 shedding (% CD16LOW) in the three groups studied. Both markers of neutrophil apoptosis increased significantly with culture time in all groups, indicating the occurrence of progressive neutrophil apoptosis (Fig 1). Differences between groups were not statistically significant. Thus, overall our results indicated that neutrophil apoptosis was occurring at the same pace in the three groups of patients studied.



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  		Figure 1. Mean ± SEM values of annexin V binding (percentage of positive cells) [left, A] and CD16 shedding (% CD16 LOW) [right, B] determined in circulating neutrophils obtained from patients with COPD ({blacktriangleup}), smokers with normal lung function (•), and healthy nonsmokers ({blacksquare}) after 2, 6, and 24 h of culture.

 
Surface Expression of Adhesion Molecules
Figure 2 presents the surface expression of Mac-1 (CD11b) and L-selectin (CD62L) during the apoptotic process in the three groups. Mac-1 expression decreased significantly with culture time in all groups (Fig 2, left, A). Interestingly, however, patients with COPD showed higher values at 6 and 24 h than healthy nonsmokers or smokers with normal lung function (Fig 2, left, A). Likewise, the expression of L-selectin (CD62L) decreased significantly with culture time in the three groups studied (Fig 2, right, B). Again, however, patients with COPD showed significantly different values at 2, 6, and 24 h than nonsmokers or smokers with normal lung function (Fig 2, right, B).



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  		Figure 2. Surface expression of CD11b (Mac-1) [left, A] and L-selectin (CD62L) [right, B] (mean ± SEM) determined in circulating apoptotic neutrophils obtained from patients with COPD ({blacktriangleup}), smokers with normal lung function (•), and healthy nonsmokers ({blacksquare}) after 2, 6, and 24 h of culture.

 

   Discussion
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Conclusions
 References
 
This study investigated potential alterations in the process of neutrophil apoptosis in COPD patients that may eventually contribute to the characteristic neutrophil accumulation described in the lungs of these patients.220 The main finding of our study was that, compared to smokers with normal lung function and never-smokers, circulating neutrophils harvested from patients with COPD show similar dynamics of apoptosis in culture, but a differential expression of two adhesion molecules (Mac-1 [CD11b] and L-selectin [CD62L]) throughout the process. The potential pathogenic implications of these observations are discussed below.

Previous Studies
It is now accepted that COPD is characterized by an abnormal inflammatory response to respiratory pollutants, mostly tobacco smoking.1 Neutrophils are found in increased concentrations in the lungs of patients with COPD.220 The mechanisms underlying this neutrophilic infiltration in COPD patients are unclear. In theory, this may be due to enhanced neutrophil recruitment and/or delayed tissue clearance. Several previous studies45 have shown an increased expression of surface adhesion molecules, which can facilitate the former. In contrast, to our knowledge, no previous study has investigated the latter in COPD patients. Yet, a dysregulation of neutrophil apoptosis has been reported in patients with a variety of other inflammatory conditions.141516

Interpretation of Findings
We found that, quantitatively, neutrophil apoptosis proceeded at similar rates in the three groups of individuals studied (Fig 1). Qualitatively, however, there were significant differences between groups because the surface expression of Mac-1 (CD11b) was higher, and that of L-selectin (CD62L) lower, in neutrophils harvested from COPD patients throughout the apoptotic process (Fig 2). These findings can have potentially relevant pathogenic implications for the following reasons. First, the initiation of the apoptotic program in neutrophils is normally linked with an impairment of their function that is aimed at limiting their ability to damage the tissue during this process.2122 Our observations are compatible with a higher activation status during the apoptotic process, because neutrophils from COPD patients showed abnormal expression of Mac-1 (CD11b) and L-selectin (CD62L).23 This suggests that neutrophils still may contribute to tissue damage in COPD patients even during apoptosis. Second, the increased surface expression of Mac-1 may alter the ability of the macrophage to recognize and engulf (thus, inactivate) an apoptotic neutrophil,2425 thus increasing their potential for tissue damage, because nonengulfed neutrophils will eventually liberate their proinflammatory content.26

Potential Limitations
Our study has some limitations that deserve comment. First, we studied the process of neutrophil apoptosis in vitro (not in vivo), in circulating neutrophils (not in neutrophils isolated from the lungs of these patients). This was due to the technical difficulties involved in trying to assess the dynamics of apoptosis in vivo and/or to harvest lung neutrophils. It is clear, therefore, that our observations cannot be readily extrapolated to what is actually occurring in the lung parenchyma of patients with COPD. However, we believe that our results provide enough scientific background to address these aspects in future studies.


   Conclusions
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
References
 
We found that, quantitatively, the rate of neutrophil apoptosis in vitro is not abnormal in COPD patients compared to healthy smokers or never-smokers. However, our study has identified qualitative differences in the surface expression of several adhesion molecules during the apoptotic process that can contribute to the perpetuation of the inflammatory state that characterizes COPD. The precise relevance of these observations in vivo requires further studies aimed at investigating neutrophil apoptosis after migration across an endothelial monolayer and the ability of lung macrophages to phagocytize apoptotic neutrophils in COPD patients.


   Acknowledgements
 
The authors thank C. Erasmo and M. Bosch, for their technical collaboration during the study, and Dr. A. De la Peña (Unidad de Investigación) for his help with the statistical analysis.


   Footnotes
 
Supported, in part, by Sociedad Española de Neumología y Cirucía Torácica, Associació Balear per L’estudi de las Malaltios Respiratorios. Red Respira (RTIC 0311, Fondo de Investigacioues Sanitarias, Instituto de Sahid Carlos III), and Fondo de Investigaciones Sanitarias grant No. 01/0830.

Received for publication April 28, 2003. Accepted for publication October 2, 2003.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusions
 References
 

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