Abstract
Alveolar macrophages (AM) participate actively in the inflammatory response that characterises chronic obstructive pulmonary disease (COPD). The present study investigated potential changes in AM phenotypes in patients with COPD.
Using flow cytometry, the surface expression of receptors implicated in phagocytosis (CD44, CD36, CD51, CD61, CD14), antigen-presenting capacity (human leukocyte antigen (HLA)-DR), costimulatory molecules (CD80, CD86, CD40) and complement receptor type 3 were assessed in AM from 18 patients with COPD, 14 smokers with normal lung function and nine nonsmokers.
When compared to smokers with normal lung function and nonsmokers, the surface expression of HLA-DR and CD80 was lower in AM of patients with COPD. In addition, these patients had a higher percentage of AM with a low level surface expression of CD44. There did not appear to be any difference in the other receptors studied in AM between the three groups. The expression of all these receptors in peripheral blood monocytes also did not differ between groups.
In conclusion, these observations suggest that the cell-mediated immune function of alveolar macrophages can be reduced in chronic obstructive pulmonary disease, and that this is a local rather than a systemic event.
Tobacco smoking is the main risk factor for chronic obstructive pulmonary disease (COPD). 1. Yet, for reasons still not fully understood, only a percentage of smokers develop COPD. Given that an abnormal inflammatory response to smoking is a hallmark of COPD 1, it is plausible that differences in the mechanisms that control the initiation and/or resolution of this inflammatory response may explain the susceptibility to develop COPD exhibited by some smokers 2.
Alveolar macrophages (AM) play a key role in both the initiation and resolution of the inflammatory response in the lower human respiratory tract 3. AM have the ability to change their surface phenotype 4, 5 and functional properties in response to exogenous stimuli and/or pathological processes in the lung parenchyma 6, 7. Some surface receptors associated with host defence and inflammation act as good markers of macrophage functional activation and can be modified by different inflammatory signals occurring in a variety of lung diseases, including sarcoidosis 8, pulmonary fibrosis 7, allergic airway inflammation 9, 10, and pulmonary tuberculosis 6. Examples of these receptors are those implicated in phagocytosis (CD44, CD36, CD51, CD61, CD14), antigen-presenting capacity (human leukocyte antigen (HLA)-DR), or several costimulatory molecules (CD80, CD86, CD40) or complement receptor type 3 (CD11b).
Increasing evidence implicates AM in the inflammatory response that characterises COPD 11, 12. In patients with COPD, AM release more matrix metalloproteinase-9 13 and interleukin-8 14, and less tissue inhibitor of metalloproteinase-1 13, 15 and transforming growth factor-β 1 15 than healthy smokers, and also have a limited ability to phagocytose apoptotic cells 16. Yet, to date their surface phenotype has not been characterised although several previous studies have investigated the influence of tobacco smoking upon AM surface phenotype 17–24.
To get a better insight into the inflammatory response of COPD, the present study sought to compare the pattern of cell surface receptor expression of AM in patients with COPD, smokers with normal lung function and nonsmokers. Peripheral blood monocytes (PBM) are the circulating precursors of AM 25. To ascertain whether potential differences of AM phenotype could also be determined in the systemic circulation, the pattern of surface receptor expression of PBM in the same individuals was studied. This may help to separate the effects derived from changes in the local pulmonary micro-environment (expected to induce phenotypic differences in AM only), from those that may potentially reflect a different genetic background (expected to induce phenotypic variance in both AM and PBM).
METHODS
Subjects and ethics
In total the following patients were recruited from the outpatient clinic of the Hospital Universitari Son Dureta, Palma de Mallorca, Spain: 18 patients with COPD (seven current smokers, 11 ex-smokers), as defined by Global Institute for Chronic Obstructive Lung Disease (GOLD) criteria (forced expiratory volume in one second (FEV1) <70% predicted) 1; 14 smokers (seven current smokers, seven ex-smokers) without chronic bronchitis or dyspnoea and with normal lung function (FEV1 >80% pred) 1; and nine nonsmokers. All required bronchoscopic evaluation of a solitary pulmonary nodule or haemoptysis. All subjects were free of any significant disease, including asthma, acute respiratory tract infections, bronchiectasis, interstitial lung diseases, and cardiac, hepatic or renal failure. All patients with COPD were clinically stable and had not experienced any exacerbations for ≥3 months preceding inclusion in the study. All were treated with inhaled bronchodilators. Four patients were treated with inhaled steroids, but none received oral steroids. Smokers and nonsmokers were unmedicated. Current and ex-smokers had a smoking history of >20 pack-yrs. Ex-smokers with normal lung function stopped smoking >3 months previously. Current smokers refrained from smoking for 12 h before bronchoscopy. All participants signed an informed consent. The ethics committee of Hospital Universitari Son Dureta (Palma de Mallorca, Spain) approved the study protocol.
Bronchoscopy
Fibreoptic bronchoscopy and bronchoalveolar lavage (BAL) were performed according to standard techniques 26. To investigate the presence of airways colonisation, which can potentially influence the results of the study, bronchial secretions were obtained using a protected specimen brush 27 and were found to be culture negative in all cases. Likewise, to avoid the potential effect of acute smoking, current smokers refrained from smoking for 12 h before bronchoscopy. This was confirmed by an exhaled CO concentration <10 ppb in all cases.
Preparation of cell suspensions
BAL samples were processed under sterile conditions within 1 h of collection and maintained at 4°C until analysis. Cells were recovered from BAL fluid by centrifugation (400×g for 10 min at 4°C), and suspended at a concentration of 1×106 cells·mL−1 in RPMI-1640 medium containing 10% foetal calf serum, 2 mM l-glutamine, 25 U·mL−1 penicillin and 25 μg·mL−1 streptomycin. Total cell counts were determined in a haemocytometer by light microscopy. Cell viability, as determined by trypan blue exclusion, was always >96%. Differential cell counts were performed by counting 500 cells on cytocentrifuge slides (Cytospin 2; Shandon Southern Prod., Ltd, Runcorn, UK) stained with Haemacolor (Merck KgaA, Darmstadt, Germany).
Blood samples were obtained by puncture of a peripheral vein. Ficoll-Hypaque gradient centrifugation (Pharmacia, Upsala, Sweden) was used to separate peripheral blood mononuclear cells, which were washed twice and suspended at a concentration of 1×106 cells·mL−1 in RPMI-1640 medium containing 10% foetal calf serum, 2 mM l-glutamine, 25 U·mL−1 penicillin and 25 μg·mL−1 streptomycin.
Inmunofluorescence labelling and flow cytometric analysis
AM and peripheral blood mononuclear cells were labelled according to the same protocol within 1 h of sampling. Of the cell suspensions (1×106 cells·mL−1), 100 μL was mixed with 10 μL of each FITC/Pe-labelled monoclonal antibody against the different surface receptors studied (Immunotech, Marseille, France). These included those implicated in phagocytosis (CD44: clone J.173; CD36: clone FA6.152; CD51: clone AMF7; CD61: clone SZ21; CD14: clone RM052), antigen-presenting capacity (HLA-DR: clone Immu-357), several costimulatory molecules (CD80: clone MAB 104; CD86: HA5.2B7; CD40: clone MAB89) and complement receptor type 3 (CD11b: clone Bear 1). Additionally, one 100-μL aliquot of AM suspension was double labelled with CD44 and HLA-DR for further identification of AM subpopulations expressing high or low surface levels of CD44 (CD44 high+ and CD44 low+, respectively). Surface expression of CD44 on AM was demonstrated by gating HLA-DR+ cells, on which the percentages of AM expressing CD44 low+ were estimated 28. Finally, peripheral blood mononuclear cells were double labelled with 10 μL of CD14 to identify PBM.
Following incubation in the dark at 4°C for 30 min and two washes in PBS, cells were re-suspended in 0.5 mL of PBS and analysed immediately in a flow cytometer (Coulter Epics XL.MCL; Coulter Corporation, Miami, FL, USA). AM were identified by gating on cells with high autofluorescence in the FITC channel, and typical forward and side-scatter characteristics. PBM were identified by gating on cells CD14+. Fluorescence histograms were generated using a three-log scale and were expressed in units of mean fluorescence intensity. The immunoglobulin (Ig)-G control values for mean fluorescence intensity (FITC/Pe IgG1, clone 679.1Mc7; Immunotech, Marseille, France) were subtracted from that of each specific monoclonal antibody. To generate each histogram 10,000 AM or 5,000 PBM were analysed.
Statistical analysis
Results are presented as mean±sem. Nonparametric statistics (Kruskal-Wallis test and Mann-Whitney U-test) were used to assess the statistical significance of differences between the groups. A p-value <0.05 was considered significant.
RESULTS
Anthropometric and lung function data
All subjects were matched for age and smoking history (table 1⇓). No significant differences were found in results obtained in current smokers and ex-smokers. According to GOLD, patients with COPD showed moderate airflow obstruction (table 1⇓) 1. By definition, spirometry was normal in smokers with normal lung function and in nonsmokers.
Clinical and lung function data of all participants
BAL cell count
Total cell count was higher in patients with COPD (1.7±0.3×105·mL−1) and smokers with normal lung function (2.3±0.4×105·mL−1) than in nonsmokers (0.5±0.1×105·mL−1; p<0.05). Differential cell counts were not different between groups (table 2⇓).
Total and differential cell counts in bronchoalveolar lavage fluid
PBM surface phenotype
The expression of the different surface receptors studied in PBM was similar in the three subject groups (table 3⇓).
Mean fluorescence intensity values of the surface receptors studied in peripheral blood monocytes (PBM) and alveolar macrophages (AM)
AM surface phenotype
The expression of most of the surface receptors studied on AM did not differ between the three subject groups (table 3⇑). However, the expression of HLA-DR and CD80 was lower (p<0.01) in patients with COPD (fig. 1⇓). The analysis of AM subpopulations (fig. 2⇓) showed that the percentage of AM with CD44 low+ was increased in patients with COPD (p<0.001; fig. 3⇓).
Surface expression of a) human leukocyte antigen (HLA)-DR and b) CD80 in alveolar macrophages obtained from healthy nonsmokers, smokers with normal lung function and patients with chronic obstructive pulmonary disease (COPD). #: p<0.02; ¶: p = 0.002; +: p = 0.003; §: p = 0.006.
Representative characterisation of alveolar macrophages (AM) in bronchoalveolar fluid from patients with a) chronic obstructive pulmonary disease and b) smokers with normal lung function. Dual expression of CD44 and human leukocyte antigen (HLA)-DR were analysed by two-colour flow cytometry; squares indicate AM expressing low levels of CD44.
Percentage of alveolar macrophages expressing low levels of CD44 (CD44 low+) in nonsmokers, smokers with normal lung function and patients with chronic obstructive pulmonary disease (COPD). ***: p<0.001.
Comparison of PBM and AM surface phenotype
The expression of complement receptor type 3 (CD11b) and that of several receptors implicated in phagocytosis (CD36, CD61 and CD14) was higher in PBM than in AM in all groups (table 3⇑). In contrast, the expression of costimulatory molecules (CD80, CD86 and CD40) was higher in AM, in the three groups studied. These differences reflect the process of maturation of PBM into AM 25, 30, 31.
DISCUSSION
Normal PBM migrate to the lungs and mature into AM 25. AM are the major immunocompetent cell population in the lower human respiratory tract, and are likely to play a key role in the initiation, modulation and eventual termination of the inflammatory response that characterises COPD 32. The different functional states of AM are mirrored by the expression of different receptors on their surface, including those implicated in phagocytosis (CD44, CD36, CD51, CD61, CD14), antigen-presenting capacity (HLA-DR), several costimulatory molecules (CD80, CD86, CD40) and complement receptor type 3 (CD11b). The current authors hypothesised that, compared with smokers with normal lung function and nonsmokers, AM in patients with COPD may present a specific surface phenotype, which may eventually contribute to an enhanced response to tobacco smoking and/or to limit the ability to stop the response once the insult has disappeared. To explore this hypothesis, the surface phenotype of AM (and PBM) was compared in these three groups of individuals. It was found that the surface expression of the majority of receptors studied was similar in all three groups. However, there were some notable exceptions. First, the expression of HLA-DR and CD80 on AM was reduced in patients with COPD and, secondly, the subpopulation of AM expressing CD44 low+ was enhanced in the same patients.
Macrophages function as antigen-presenting cells and thereby orchestrate the activation of different subsets of lymphocytes. HLA-DR regulates antigen presentation by AM, and their expression on the surface of monocytes/macrophages is strongly related to the activation state of these cells 10. Hence, reduced expression of HLA-DR on AM is linked with a hypo-activation state 33 and may promote immune tolerance 34, 35. Moreover, CD80 plays an important role in providing costimulatory signals required for the development of antigen-specific immune responses 36. Thus, the reduced expression of HLA-DR and CD80 found in AM of COPD patients suggests that the cell-mediated immune function of AM may be abnormal in these patients. This may favour bronchial colonisation by interfering with the recognition of the antigen complex on the surface of AM by CD4+ T-helper lymphocytes 37. The fact that the expression of HLA-DR and CD80 on PBM was similar in the three groups of subjects studied suggests that the different phenotypes observed in AM occur in response to changes in the local pulmonary milieu and rules out the possibility of a different genetic background in these patients.
CD44 is a transmembrane adhesion receptor and the major cell surface receptor for the glycosaminoglycan hyaluronan 38. As such, CD44 plays an important role in the clearance of hyaluronan, which accumulates at sites of inflammation and tissue injury, and has pro-inflammatory functions 39. In the normal lung, AM are the major cell type known to internalise hyaluronan for degradation, and CD44 participates in this process 40. Moreover, CD44 ligation on macrophages selectively promotes the uptake of apoptotic neutrophils in vitro 41. Therefore, overall CD44 plays a major role in the resolution of lung inflammation 42. The current study found that the percentage of AM expressing CD44 low+ was higher in patients with COPD than in smokers with normal lung function and nonsmokers. This may determine a decreased hyaluronan-binding capacity 28 and, therefore, limit the ability of AM in patients with COPD to clear apoptotic neutrophils 41–43, and contribute to perpetuate lung inflammation. Yet, because of the the cross-sectional design of the current study, it cannot be established whether these alterations are involved in the primary pathogenesis of the disease or are a consequence of it.
In conclusion, the current study shows that the alveolar macrophages in patients with chronic obstructive pulmonary disease exhibit a specific surface phenotype, characterised by reduced expression of two receptors involved in antigen presentation (human leukocyte antigen-DR and CD80) and one receptor involved in the termination of the inflammatory response (CD44). These observations highlight two previously undescribed alterations that may contribute to the abnormal inflammatory response that characterises chronic obstructive pulmonary disease.
Acknowledgments
The authors would like to thank the participating individuals for their collaboration in the study; and F. Bauzà, M. Bosch, A. Noguera and M. López for their help during the study.
- Received May 27, 2004.
- Accepted December 3, 2004.
- © ERS Journals Ltd
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