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Original Research: COPD |

Distribution of T-Cell Subsets in BAL Fluid of Patients With Mild to Moderate COPD Depends on Current Smoking Status and Not Airway ObstructionBAL T Cells in COPD and Smoking FREE TO VIEW

Helena Forsslund, MSc; Mikael Mikko, PhD; Reza Karimi, MD; Johan Grunewald, MD, PhD; Åsa M. Wheelock, PhD; Jan Wahlström, PhD; C. Magnus Sköld, MD, PhD
Author and Funding Information

From the Respiratory Medicine Unit, Department of Medicine Solna and Centre for Molecular Medicine, Karolinska Institutet, Karolinska University Hospital, Solna, Stockholm, Sweden.

Correspondence to: Helena Forsslund, MSc, Lung Research Laboratory L4:01, Respiratory Medicine Unit, Department of Medicine Solna and CMM, Karolinska Institutet and Karolinska University Hospital, Solna, Stockholm 171 76, Sweden; e-mail: Helena.Forsslund@ki.se


Parts of the data from this article were presented at the European Respiratory Society Congress, September 24-28, 2011, Amsterdam, The Netherlands and published in abstract form (Forsslund H, Mikko M, Grunewald J, Wheelock ÅM, Wahlström J, Sköld CM. Characterization of lymphocyte subsets in the lungs of smokers and patients with COPD. Eur Respir J. 2011;38(suppl 55):P1834).

Funding/Support: Financial support for this study was provided through the Swedish Heart-Lung Foundation, the King Oscar II Jubilee Foundation, the Mats Kleeberg Foundation, King Gustaf V’s and Queen Victoria’s Freemasons’ Foundation, the Hesselmans Foundation, Swedish Governmental Agency for Innovation Systems (VINNOVA), the Swedish Foundation for Strategic Research (SSF), European Union (EU) Fp6 Marie Curie International Reintegration Grant (IRF), and the Karolinska Institutet and The Swedish Research Council.

Reproduction of this article is prohibited without written permission from the American College of Chest Physicians. See online for more details.


Chest. 2014;145(4):711-722. doi:10.1378/chest.13-0873
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Background:  COPD is characterized by chronic inflammation. CD8+ T cells and CD4+ T cells have both been implicated in the inflammatory response. We investigated whether the lymphocyte and T-cell subpopulations in BAL differ between patients with COPD who are current smokers and those who are ex-smokers.

Methods:  Forty never smokers, 40 smokers with normal lung function, and 38 patients with COPD, GOLD (Global Initiative for Chronic Obstructive Pulmonary Disease) stage I-II (27 smokers and 11 ex-smokers) underwent BAL. Using flow cytometry, cells were analyzed from BAL and blood for T-cell subsets, B cells, natural killer cells, and natural killer T (NKT)-like cells. The differentiation status of CD4+ T cells was also determined.

Results:  Smokers with or without COPD had higher percentages of CD8+ T cells and NKT-like cells in BAL than did never smokers and ex-smokers with COPD. Most of the NKT-like cells were CD8+. In contrast, the percentages of CD4+ T cells were lower in the smoking than in the nonsmoking groups. In blood, the frequency of CD4+ T cells was increased in the two smoking groups. Current smokers also had increased numbers of activated (CD69+) naive and effector CD4+ T cells in BAL compared with nonsmokers, particularly in patients with COPD. In male smokers with COPD, the percentage of CD8+ T cells in BAL positively correlated with the number of cigarettes per day.

Conclusions:  Current smoking status has a greater impact than airway obstruction on the distribution of T-cell subsets in BAL of patients with mild to moderate COPD. This fact must be considered when the role of T cells in COPD is evaluated. Our results stress the importance of subgrouping patients with COPD in terms of smoking.

Figures in this Article

COPD is the fourth-leading cause of death worldwide.1 The disease is characterized by small airway remodeling and tissue destruction, which are most frequently consequences of an inflammatory response to inhaled smoke. The mechanisms leading to the initiation and persistence of this smoke-induced inflammation are not fully understood but are important to outline to prevent and treat the disease.

Cigarette smoking per se leads to a substantial local inflammation in the lungs,2 and it is, therefore, essential to distinguish changes induced and maintained by cigarette smoke from those related to disease. When evaluating ex-smokers with COPD, it is also important to consider smoking history, in particular the time elapsed since smoking cessation, because the normalization process after quitting smoking leads to a reduction in the number of inflammatory cells in the lungs3 but a persistence of airway inflammation in patients with COPD.4,5

Recruitment of lymphocytes is believed to play a central role in the pathogenesis of COPD.68 Previous studies have shown that the number of CD8+ T cells is increased in both central and peripheral airways, as well as in the lung parenchyma, in patients with COPD and correlates negatively with the degree of airflow obstruction.9,10 Altogether, these findings suggest a key role for CD8+ T cells in COPD-related tissue destruction. CD4+ T cells are also likely to play a role in COPD, particularly in patients with severe disease, because these cells are increased in number in the lung tissue of patients with severe emphysema.11

To better understand the role of T cells in relation to smoking and in the pathogenesis of COPD, it is indispensable to map out the different T-cell phenotypes and their local and systemic distribution in COPD compared with healthy subjects. T-cell maturation can be divided into four stages, which can be identified based on their expression of the surface markers CD45RA and CD27. By combining these markers, it is possible to discriminate naive (CD27+CD45RA+), central memory (CD27+CD45RA), effector memory (CD27CD45RA), and effector (CD27CD45RA+) subpopulations.1215

In addition to CD8+ and CD4+ T cells, other lymphocyte subsets may be involved in smoke-induced inflammation. Natural killer (NK) cells are large granular lymphocytes that express CD16, CD56, or both. They are capable of destroying target cells without prior sensitization and play an important role in immune surveillance against tumors and resistance against viral infections16 but may contribute to inflammation in many other settings. T cells can also express the NK markers CD16 and CD56. These CD3+/CD16+ and/or CD56+ cells are generally referred to as natural killer T (NKT)-like cells and display features of both T cells and NK cells.17

In this study, we addressed major lymphocyte subsets and the differentiation status of T cells, both locally and systemically, in relation to smoking. We hypothesized that not only smoking history, expressed as pack-years, but also current smoking status, may determine the distribution of T-cell subsets in the lung, including subpopulations reflecting maturation. To test this hypothesis, we investigated T cells in BAL and blood from both current smokers with COPD and ex-smokers with COPD and compared them with never smokers and smokers with normal lung function.

Study Subjects and Patients

The study was carried out on subjects from the Karolinska COPD and Smoking from an OMIC Perspective (COSMIC) cohort at the Karolinska University Hospital Solna, Karolinska Institutet, Stockholm, Sweden. The aim of the COSMIC study is to investigate and integrate several aspects of COPD and smoking through imaging, transcriptomics, proteomics, metabolomics, and lymphocyte profiling in the context of clinical phenotypes.1820 A total of 40 never smokers, 40 smokers with normal lung function (hereafter referred to as “smokers”), and 38 patients with COPD were recruited with the intent to collect peripheral blood (PB) and BAL (Table 1). The patients and control subjects were recruited from among individuals performing spirometry during “The World Spirometry Day,” through advertisements in the daily press and via primary care centers. The majority of the patients with COPD were smokers who turned out to have an obstructive spirometry on screening. Three patients and one never smoker did not have BAL because of clinical constraints, but PB was still used for analysis. Of the patients with COPD, 27 were current smokers, and 11 were ex-smokers who had quit smoking a minimum of 2 years before entering the study. Ten of the smokers, seven of the smokers with COPD, and two of the ex-smokers with COPD fulfilled the criteria for chronic bronchitis.21 Spirometry (Jaeger Masterscope-PC; CareFusion) was performed, and the patients with COPD had a postbronchodilator FEV1/FVC < 0.7 and an FEV1 50% to 100% predicted (ie, GOLD [Global Initiative for Chronic Obstructive Pulmonary Disease] stage I and II).22 The distribution of patients according to the updated COPD classification1 is shown in Table 1. None of the subjects had had any exacerbations during the previous 3 months. Patients with a history of allergy or asthma were excluded, as were patients using oral or inhaled corticosteroids. In vitro screenings for the presence of specific IgE antibodies (Phadiatop; Pharmacia Corp) were negative. Reversibility was tested after inhalation of two doses of 0.25 mg terbutaline (Bricanyl; Turbuhaler; AstraZeneca). Patients with COPD had significantly more dyspnea and ex-smokers with COPD had lower physical domain scores, as assessed by the chronic respiratory disease standardized questionnaire addressing quality of life23 (Table 1). All participants provided written informed consent, and the study was approved by the regional ethics committee in Stockholm on October 26, 2006 (ref: 2006/959-31/1).

Table Graphic Jump Location
Table 1 —Characteristics and Lung Function Data of Never Smokers, Smokers, and Patients With COPD

Data are presented as median (range) unless indicated otherwise. CRQ = Chronic Respiratory Disease Questionnaire; Dlco = diffusion capacity of the lung for carbon monoxide; GOLD = Global Initiative for Chronic Obstructive Pulmonary Disease; NA = not applicable; RV = residual volume.

a 

P < .05 when compared with smokers.

b 

P < .001 when compared with never smokers.

c 

P < .001 when compared with smokers.

d 

P < .01 when compared with never smokers.

e 

P < .05 when compared with never smokers.

BAL Procedure and Processing of BAL Fluid and PB

Bronchoscopy and BAL were performed according to a standard protocol at our clinic. For details, see e-Appendix 1.

Macrophage Depletion

Prior to monoclonal antibody staining, macrophage depletion from the BAL cells was performed as described previously.24 For details, see e-Appendix 1.

Measurement of Systemic Inflammatory Markers

Serum acute-phase proteins C-reactive protein, α1-antitrypsin, orosmucoid, serum-albumin, and haptoglobin were measured by routine methods. Measurements were done at the laboratory of Karolinska University Hospital, Solna, Sweden.

Immunofluorescent Surface Staining and Flow Cytometry

Monoclonal antibodies in two different panels were used to characterize the major lymphocyte subsets and the T-cell differentiation subsets in BAL and PB (e-Table 1). For identification of all NK and NKT cells, a combination of phycoerythrin-conjugated monoclonal antibodies against both CD16 and CD56 was used (BD Medical). BAL and blood cells were stained with surface markers as described in e-Appendix 1.

Lymphocyte subsets in BAL and PB were analyzed using eight-color flow cytometry (FACSCanto II; BD Medical). Data were processed in FACSDiva 6.1.2 (BD Medical). Flow cytometric data were excluded from the data analysis if fewer than 50 events were detected in the final gate.

Statistical Analyses

Data are presented as median (range) unless otherwise stated. Differences between groups were analyzed using Kruskal-Wallis one-way analysis of variance followed by Dunn’s multiple comparison tests. A P value < .05 was considered statistically significant. Correlation analyses were assessed with the Spearman rank correlation test, and the resulting P values were adjusted for multiple testing using the false discovery rate (FDR) according to Benjamini and Hochberg.25 An FDR < 0.05 was considered statistically significant. Statistical comparisons were performed and graphs were prepared using GraphPad Prism software, version 5.02 (GraphPad Software).

We studied two groups of clinically stable patients with COPD with GOLD stage I-II (27 current smokers and 11 ex-smokers) and compared them with 40 never smokers and 40 smokers.

BAL Cell Data and Markers of Systemic Inflammation

The characteristics of the BAL, including BAL fluid recovery measured as a percentage of instilled fluid, the percentages of viable BAL cells, the cell concentration, and differential counts, are shown in Table 2. The serum levels of C-reactive protein, α1-antitrypsin, orosmucoid, serum-albumin, and haptoglobin are reported in e-Appendix 1 and in e-Table 2.

Table Graphic Jump Location
Table 2 —BAL Characteristics of Never Smokers, Smokers, Smokers With COPD, and Ex-Smokers With COPD

Data are presented as median (range) unless indicated otherwise.

a 

P < .001 when compared with never smokers.

b 

P < .01 when compared with smokers.

c 

P < .01 when compared with never smokers.

d 

P < .001 when compared with smokers.

e 

P < .001 when compared with smokers with COPD.

f 

P < .01 when compared with smokers with COPD.

g 

P < .05 when compared with smokers with COPD.

h 

P < .05 when compared with smokers.

Lymphocyte Characterization in BAL and PB

The relative frequencies of CD4+ and CD8+ T cells, B cells, NK cells, and NKT-like cells in BAL and PB were characterized with a multicolor test (e-Table 1).

CD8+ T Cells:

The percentage of CD8+ T cells was significantly higher in BAL from smokers and smokers with COPD compared with never smokers (P < .001 and P < .01, respectively), whereas there was a lower percentage of CD8+ T cells in BAL from ex-smokers with COPD compared with both smoking groups (P < .01 for both) (Fig 1A). In PB, the inverse trend was seen: Smokers and smokers with COPD had lower median percentages of CD8+ T cells compared with never smokers.

Figure Jump LinkFigure 1. A-F, Lymphocyte characterization in BAL and PB. Cells were stained with a multicolor test to characterize the lymphocyte cell types (A) CD8+ T cells, (B) CD4+ T cells, (C) the CD4/CD8 ratio, (D) natural killer (NK) cells, (E) natural killer T (NKT)-like cells, and (F) B cells, in NSs, Ss, CSs, and CEs. G-I, Representative flow cytometric dot plots of (G) CD4+ and CD8+ among CD3+ T cells, (H) NK cells, identified as CD3CD56+/CD16+ cells, and NKT-like cells, identified as CD3+CD56+/CD16+ cells, among CD45+ cells, and (I) B cells, identified as CD3CD19+ cells, among CD45+ cells. *P < .05; **P < .01; ***P < .001. CE = ex-smoker with COPD; CS = smoker with COPD; NS = never smoker; PB = peripheral blood; S = smoker.Grahic Jump Location
CD4+ T Cells:

The proportion of CD4+ T cells was significantly reduced in BAL from both smokers and smokers with COPD compared with never smokers (P < .001 for both) and ex-smokers with COPD (P < .001 and P < .01, respectively) (Fig 1B). In contrast, the proportion of CD4+ T cells was increased in PB from both smoking groups compared with never smokers (P < .05 for both).

CD4/CD8 Ratios:

The CD4/CD8 ratio in BAL from smokers and smokers with COPD was significantly lower than that of never smokers (P < .001 for both comparisons) and that of ex-smokers with COPD (P < .01 for both comparisons) (Fig 1C). In PB, no significant differences in the CD4/CD8 ratio was observed among the groups, even though there was a trend toward increased CD4/CD8 ratios in smokers, smokers with COPD, and ex-smokers with COPD compared with never smokers.

NK Cells, NKT-Like Cells, and B Cells:

The proportion of NK cells (CD3 and CD16+ and/or CD56+) was significantly higher in BAL from smokers compared with both never smokers and ex-smokers with COPD (P < .001 and P < .05, respectively) (Fig 1D). A nonsignificant decrease in the percentage of NK cells was seen in PB from both groups of smokers compared with never smokers and ex-smokers with COPD. The percentage of NKT-like cells (CD3+ and CD16+ and/or CD56+) was higher in BAL from both smokers and smokers with COPD compared with never smokers (P < .001 for both) and ex-smokers with COPD (P < .01 and P < .05, respectively) (Fig 1E). A significant decrease in the percentage of NKT-like cells was seen in PB from smokers compared with never smokers (P < .05). B cells were found in low frequencies in BAL but to a much higher extent in PB. No differences in frequency were found among the groups (Fig 1F).

NKT-Like Cell Subsets:

Four NKT-like cell subpopulations, CD4+, CD8+, CD4CD8, and CD4+CD8+, were identified in both BAL and PB. In BAL, the percentage of CD8+ NKT-like cells was significantly higher in smokers than in both never smokers and ex-smokers with COPD (P < .001 and P < .01, respectively) (Fig 2A). Consequently, the percentage of CD4+ NKT-like cells was significantly lower in smokers (P < .001 and P < .05, respectively) (Fig 2B). No significant differences were seen among the CD4CD8 NKT-like subpopulation in BAL (data not shown), whereas CD4+CD8+ were too few to determine. In PB, smokers with COPD had a significantly lower percentage of CD4+ NKT-like cells compared with smokers (P < .05). No other differences in PB among the groups were observed (data not shown).

Figure Jump LinkFigure 2. A-D, Proportions of (A) CD8+ cells and (B) CD4+ cells, among CD16+ and/or CD56+ T cells (ie, NKT-like cells), and CD16+ and/or CD56+ cells among (C) CD8+ T cells and (D) CD4+ T cells in BAL. *P < .05; **P < .01; ***P < .001. See Figure 1 legend for expansion of abbreviations.Grahic Jump Location
NKT-Like Cells Among CD4+ and CD8+ T Cells:

Both groups of smokers had higher proportions of NKT-like cells among CD8+ T cells in BAL compared with never smokers (P < .001 for both) and ex-smokers with COPD (P < .05 for both) (Fig 2C). An increase was also seen among CD4+ T cells in BAL from smokers and smokers with COPD compared with never smokers (P < .01 and P < .05, respectively) (Fig 2D). No differences were seen in PB (data not shown).

CD27 and CD45RA Subsets

In BAL, effector memory cells were the dominating subset among CD4+ T cells, followed by central memory cells. In contrast, the largest populations among PB CD4+ T cells were naive and central memory T cells, and only a minority were effector memory T cells (Fig 3). Although not statistically significant, there were higher percentages of naive cells, central memory cells, and effector cells, but lower percentages of effector memory cells, in BAL from both smoking groups compared with never smokers and ex-smokers with COPD. In PB, a significant decrease in the percentage of effector CD4+ T cells was seen in smokers with COPD compared with never smokers (P < .01).

Figure Jump LinkFigure 3. A-D, Characterization of T-cell differentiation in BAL and PB CD4+ cells using CD27 and CD45RA expression. (A) CM cells, (B) naive cells, (C) EM cells, and (D) effector cells, in NSs, Ss, and patients with COPD. E-F, Representative flow cytometric dot plots of CD27 stained together with CD45RA among CD3+CD4+ T cells in (E) BAL and (F) PB. **P < .01. CM = central memory; EM = effector memory. See Figure 1 legend for expansion of other abbreviations.Grahic Jump Location

The percentage of activated cells among the four subpopulations of CD4+ T cells in BAL was determined as the percentage of each subpopulation of CD4+ T cells that expresses the early activation marker CD69 (Fig 4). Smokers had significantly higher proportions of CD69-expressing naive CD4+ T cells (P < .05), and the same tendency was observed in central memory and effector cells. Smokers with COPD had a significantly higher percentage of CD69+ naive CD4+ T cells in BAL compared with both never smokers and ex-smokers with COPD (P < .001 and P < .01, respectively) and a significantly higher percentage of CD69+ central memory and effector CD4+ T cells in BAL compared with never smokers (P < .05 and P < .01, respectively). The percentage of CD69+ cells among the four subpopulations of CD4+ T cells could not be determined in PB because of an insufficient number of events for accurate statistical comparisons. Because of difficulties in separating CD45RA-positive cells from CD45RA-negative cells among CD8+ T cells in BAL and PB, we were not able to classify the distribution of naive, effector, and memory subsets in the CD8+ T cells.

Figure Jump LinkFigure 4. A-D, Expression of the activation marker CD69 among (A) CM, (B) naive, (C) EM, and (D) effector CD4+ T cells in BAL. *P < .05; **P < .01; ***P < .001. See Figure 1 and 3 legends for expansion of abbreviations.Grahic Jump Location
Correlations Between CD8+ T Cells and Smoking

The percentage of CD8+ BAL T cells from smokers with COPD showed a positive correlation with the number of cigarettes smoked per day during the previous 6 months (rs = 0.63, P[FDR] = .04) (data not shown). This correlation was even stronger in male smokers with COPD (rs = 0.82, P [FDR] = .03) (Fig 5).

Figure Jump LinkFigure 5. Correlation between the percentage of CD8+ T cells in BAL from male smokers with COPD and the number of cigarettes smoked per day during the preceding 6 months. p[FDR] = P value corrected for multiple testing by means of false discovery rate according to Benjamini-Hochberg; rs = Spearman rank correlation coefficient.Grahic Jump Location
Correlations Between Lymphocyte Subpopulations and Other Parameters

There were no correlations between any of the measured lymphocyte subpopulations in BAL and patient characteristics, BAL characteristics, or markers of systemic inflammation (e-Table 3). Neither were there any significant differences among sexes, GOLD stage, and chronic bronchitis.

This study demonstrates that current smoking status, and not airway obstruction, determines the distribution of T-cell subsets in BAL from patients with mild to moderate COPD. In particular, increased percentages of BAL CD8+ T cells and NKT-like cells were shown among smokers with and without COPD, compared with never smokers. This increase was not seen in ex-smokers with COPD. Among the NKT-like cells, most were CD8+ in smokers, compared with never smokers and ex-smokers with COPD. Both groups of smokers had higher proportions of NKT-like cells among CD8+ T cells compared with never smokers and ex-smokers with COPD. In addition, activated (CD69+) naive, central memory, and effector CD4+ T cells were seen more commonly in BAL from smokers with COPD.

The significantly lower volumes of recovered BAL fluid from patients with COPD, both current smokers and ex-smokers, compared with both never smokers and healthy smokers, are in concordance with the findings of previous studies,2628 as were the higher percentages and concentration of macrophages, as well as the lower percentages of lymphocytes in BAL, from both smoking groups compared with both never smokers and ex-smokers with COPD.29 In the current study, we present data on lymphocyte subsets based on relative and not absolute cell numbers, because we believe that changes in relative numbers better reflect the specificity of the inflammatory response in COPD. A comparison of absolute cell numbers in BAL among study groups is more suitable when equivalent volumes of BAL are recovered. In the current study, because the concentration of lymphocytes in BAL was comparable among the studied groups, the alterations in percentages of lymphocyte subsets were also reflected in the absolute cell numbers.

Studies comparing smokers with ex-smokers are sparse. A few histopathologic studies comparing lung tissue in COPD indicate that structural changes in patients with COPD do not reverse after smoking cessation,30,31 despite an FEV1 decline.32,33 Most studies of BAL from patients with COPD have not taken current smoking status among the patients into consideration. Hodge et al34 showed that smokers with and without COPD had significantly increased percentages of CD8+ T cells, but decreased percentages of CD4+ T cells, in BAL, compared with nonsmokers. This alteration was not seen in patients with COPD who had quit smoking more than 1 year previously. Our results show that the distribution of T-cell subsets differs between smokers with COPD and ex-smokers with COPD, indicating that these changes are smoke induced and are reversible after smoking cessation, emphasizing the importance of analyzing current smokers with COPD and ex-smokers with COPD separately. The impact of current smoking status on T cells was further demonstrated by the significant positive correlation between cigarettes/day during the preceding 6 months and the percentage of CD8+ T cells in BAL in smokers with COPD, especially among men. The reason for this discrepancy between men and women in this respect is unclear.

COPD is associated with increased numbers of CD8+ T cells in the lung parenchyma and bronchial tissue.9,10,35 In previous studies, which included a limited number of patients, it was shown that smokers with and without airway obstruction exhibit an increase in CD8+ T cells and a decrease in CD4+ T cells in BAL.29,34 The current study extends these observations in larger groups of patients and control subjects. Smoking had a significant impact not only on T cells in BAL but also on T cells in the systemic circulation. Reciprocal changes, albeit of a smaller magnitude but in some cases statistically significant, were observed in PB, suggesting preferential recruitment of CD8+ T cells to the lung.

Our findings of a higher percentage of NK cells (CD3 and CD16+ and/or CD56+) in BAL from smokers compared with never smokers and a trend toward a lower percentage of NK cells in PB from both smoker groups are generally in agreement with the findings of previous studies.36,37 Currently smoking and ex-smoking patients with COPD have been shown to have higher levels of granzyme B+ NK cells in BAL with a higher cytotoxic activity compared with healthy control subjects.36 In PB, however, smokers have been shown to have a defective cytotoxic activity among NK cells.38,39 Defective peripheral NK cytotoxic activity and phagocytosis have also been shown in ex-smokers with COPD.40 Of note, CD56 expression on CD3 cells is not exclusive to NK cells; NK cells are considered part of a family of innate lymphoid cells (ILCs),41,42 and some ILC subsets have been shown to express CD56. Therefore, we cannot rule out the possibility that the CD3CD56+ cells that we refer to as NK cells include other ILCs. Because no single specific marker (or combination of a few markers) unique for ILCs has been discovered, flow cytometric identification of these cells requires a large number of antibodies for each ILC subset, which was beyond the means of this study. Furthermore, the existence of some of the ILC subsets was reported only recently.43,44 Investigations into ILCs, other than NK cells, in the human lung are sparse, but based on published data,45 such cells could be expected to be few and to make up only a minor or negligible portion of the CD3CD56+ cells in BAL.

In this study, T cells that express either or both of the NK-associated receptors CD16 and CD56 are designated NKT-like cells.46 This denomination generally refers to NKT cells with a diverse T cell receptor that recognize major histocompatibility molecules, but can in fact also include CD1d-restricted NKT cells, such as the classic type 1/invariant NKT cells.4749 We found a significant increase of CD16+, CD56+, or both T cells in BAL from both smoker groups in comparison with never smokers. In addition to previous studies showing increased relative numbers of CD3+CD56+ T cells in BAL from smokers and smokers with COPD36 and in induced sputum from smokers with COPD,50 we show that for patients with COPD the increase is likely to reverse after smoking cessation.

The smoke-induced increase in CD8+ T cells, NK cells, and NKT-like cells in BAL may increase the cytotoxic burden in the lung,34,5153 because these cells have the capacity to express granzyme B and perforin and, by a variety of mechanisms, stimulate cell death of epithelial and other structural cells.54 They may also affect other cells via cytokine secretion. Functional studies of these cell types are needed to elucidate their role in smoke-induced inflammation.

There were no significant differences in the proportions of B cells among the study groups, either in BAL or PB. Several histopathologic studies have been done on B cells in the small airways and lung parenchyma of patients with COPD55,56 but, to our knowledge, no study with published results has investigated the levels of B cells in BAL from smokers and patients with COPD.

Our results extend those of previous reports57 by providing information about central memory and effector memory cells of the BAL CD4+ population in COPD. Even though no significant differences were seen among the groups regarding the differentiation status of the CD4 T-cell subsets, either in BAL or in the periphery, the percentages of naive, effector, and central memory CD4+ T cells in BAL expressing CD69 were increased in smokers with COPD. This finding indicates that current smoking together with COPD is associated with activated T-cell subsets and that although the total CD4 number is unchanged, there may be important phenotypic changes.

A limitation to our study is that we investigated only BAL fluid and not tissue specimens. This limits our ability to draw conclusions about T-cell subsets in the airway wall or lung parenchyma. On the other hand, BAL has the advantage of providing cells in solution, which makes it possible to study multiple cellular markers.

In mild to moderate COPD, current smoking status, and not airway obstruction, has an impact on BAL cell differential counts and on the distribution of lymphocyte subsets, regarding CD4+ T cells, CD8+ T cells, NK cells, and NKT-like cells and the allocation of subsets reflecting CD4 T-cell activation. We conclude that current smoking status is crucial when the inflammatory response in COPD is evaluated. We would, therefore, like to stress the importance of subtyping patients with COPD, in particular regarding smoking habits.

Author contributions: Ms Forsslund served as principal investigator and is the guarantor of the manuscript, taking responsibility for the integrity of the data and the accuracy of the data analysis.

Ms Forsslund: contributed to the study design; collection, major analysis, and interpretation of data; drafting of the manuscript; and critical review of the manuscript, including review of the final version.

Dr Mikko: contributed to the study design; collection, analysis, and interpretation of data; and critical review of the manuscript, including review of the final version.

Dr Karimi: contributed to the collection, analysis, and interpretation of data and critical review of the manuscript, including review of the final version.

Dr Grunewald: contributed to the study design; interpretation of data; and critical review of the manuscript, including review of the final version.

Dr Wheelock: contributed to the initiation of the project; study design; and critical review of the manuscript, including review of the final version.

Dr Wahlström: contributed to the study design; analysis and interpretation of data; drafting of parts of the manuscript; and critical review of the manuscript, including review of the final version.

Dr Sköld: contributed to the initiation of the project; study design; collection, analysis, and interpretation of data; drafting of parts of the manuscript; and critical review of the manuscript, including review of the final version.

Financial/nonfinancial disclosures: The authors have reported to CHEST that no potential conflicts of interest exist with any companies/organizations whose products or services may be discussed in this article.

Role of sponsors: The sponsors had no role in the design of the study, the collection and analysis of data, or in the preparation of the manuscript.

Other contributions: The authors thank Benita Engvall, MSc; Gunnel de Forest, RN; Heléne Blomqvist; and Margitha Dahl, RN, for their excellent technical assistance.

Additional information: The e-Appendix and e-Tables can be found in the “Supplemental Materials” area of the online article.

FDR

false discovery rate

GOLD

Global Initiative for Chronic Obstructive Pulmonary Disease

ILC

innate lymphoid cell

NK

natural killer

NKT

natural killer T

PB

peripheral blood

Global strategy for the diagnosis, management, and prevention of COPD. GOLD website. http://www.goldcopd.org. Accessed November 25, 2013.
 
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Calabrese F, Giacometti C, Beghe B, et al. Marked alveolar apoptosis/proliferation imbalance in end-stage emphysema. Respir Res. 2005;6:14. [CrossRef]
 
Saetta M, Baraldo S, Corbino L, et al. CD8+ve cells in the lungs of smokers with chronic obstructive pulmonary disease. Am J Respir Crit Care Med. 1999;160(2):711-717. [CrossRef]
 
O’Shaughnessy TC, Ansari TW, Barnes NC, Jeffery PK. Inflammation in bronchial biopsies of subjects with chronic bronchitis: inverse relationship of CD8+ T lymphocytes with FEV1. Am J Respir Crit Care Med. 1997;155(3):852-857. [CrossRef]
 
Turato G, Zuin R, Miniati M, et al. Airway inflammation in severe chronic obstructive pulmonary disease: relationship with lung function and radiologic emphysema. Am J Respir Crit Care Med. 2002;166(1):105-110. [CrossRef]
 
Hamann D, Baars PA, Rep MH, et al. Phenotypic and functional separation of memory and effector human CD8+ T cells. J Exp Med. 1997;186(9):1407-1418. [CrossRef]
 
Hamann D, Roos MT, van Lier RA. Faces and phases of human CD8 T-cell development. Immunol Today. 1999;20(4):177-180. [CrossRef]
 
Hamann D, Kostense S, Wolthers KC, et al. Evidence that human CD8+CD45RA+CD27- cells are induced by antigen and evolve through extensive rounds of division. Int Immunol. 1999;11(7):1027-1033. [CrossRef]
 
Okada R, Kondo T, Matsuki F, Takata H, Takiguchi M. Phenotypic classification of human CD4+ T cell subsets and their differentiation. Int Immunol. 2008;20(9):1189-1199. [CrossRef]
 
Vivier E, Tomasello E, Baratin M, Walzer T, Ugolini S. Functions of natural killer cells. Nat Immunol. 2008;9(5):503-510. [CrossRef]
 
Peralbo E, Alonso C, Solana R. Invariant NKT and NKT-like lymphocytes: Two different T cell subsets that are differentially affected by ageing. Exp Gerontol. 2007;42(8):703-708. [CrossRef]
 
Sandberg A, Sköld CM, Grunewald J, Eklund A, Wheelock AM. Assessing recent smoking status by measuring exhaled carbon monoxide levels. PLoS ONE. 2011;6(12):e28864. [CrossRef]
 
Mikko M, Forsslund H, Cui L, et al. Increased intraepithelial (CD103+) CD8+ T cells in the airways of smokers with and without chronic obstructive pulmonary disease. Immunobiology. 2013;218(2):225-231. [CrossRef]
 
Kohler M, Sandberg A, Kjellqvist S, et al. Gender differences in the bronchoalveolar lavage cell proteome of patients with chronic obstructive pulmonary disease. J Allergy Clin Immunol. 2013;131(3):743-751. [CrossRef]
 
Definition and classification of chronic bronchitis for clinical and epidemiological purposes. A report to the Medical Research Council by their Committee on the Aetiology of Chronic Bronchitis. Lancet. 1965;1(7389):775-779.
 
Rabe KF, Hurd S, Anzueto A, et al; Global Initiative for Chronic Obstructive Lung Disease. Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease: GOLD executive summary. Am J Respir Crit Care Med. 2007;176(6):532-555. [CrossRef]
 
Guyatt G. Measuring health status in chronic airflow limitation. Eur Respir J. 1988;1(6):560-564.
 
Wong DM, Varesio L. Depletion of macrophages from heterogeneous cell populations by the use of carbonyl iron. Methods Enzymol. 1984;108:307-313.
 
Benjamini Y, Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc, B. 1995;57(1):289-300.
 
Löfdahl JM, Cederlund K, Nathell L, Eklund A, Sköld CM. Bronchoalveolar lavage in COPD: fluid recovery correlates with the degree of emphysema. Eur Respir J. 2005;25(2):275-281. [CrossRef]
 
Röpcke S, Holz O, Lauer G, et al. Repeatability of and relationship between potential COPD biomarkers in bronchoalveolar lavage, bronchial biopsies, serum, and induced sputum. PLoS ONE. 2012;7(10):e46207. [CrossRef]
 
Barceló B, Pons J, Fuster A, et al. Intracellular cytokine profile of T lymphocytes in patients with chronic obstructive pulmonary disease. Clin Exp Immunol. 2006;145(3):474-479. [CrossRef]
 
Roos-Engstrand E, Ekstrand-Hammarström B, Pourazar J, Behndig AF, Bucht A, Blomberg A. Influence of smoking cessation on airway T lymphocyte subsets in COPD. COPD. 2009;6(2):112-120. [CrossRef]
 
Wright JL, Lawson LM, Pare PD, Wiggs BJ, Kennedy S, Hogg JC. Morphology of peripheral airways in current smokers and ex-smokers. Am Rev Respir Dis. 1983;127(4):474-477.
 
Wright JL, Hobson J, Wiggs BR, Pare PD, Hogg JC. Effect of cigarette smoking on structure of the small airways. Lung. 1987;165(2):91-100. [CrossRef]
 
Fletcher C, Peto R. The natural history of chronic airflow obstruction. BMJ. 1977;1(6077):1645-1648. [CrossRef]
 
Postma DS, de Vries K, Koëter GH, Sluiter HJ. Independent influence of reversibility of air-flow obstruction and nonspecific hyperreactivity on the long-term course of lung function in chronic air-flow obstruction. Am Rev Respir Dis. 1986;134(2):276-280.
 
Hodge G, Nairn J, Holmes M, Reynolds PN, Hodge S. Increased intracellular T helper 1 proinflammatory cytokine production in peripheral blood, bronchoalveolar lavage and intraepithelial T cells of COPD subjects. Clin Exp Immunol. 2007;150(1):22-29. [CrossRef]
 
Saetta M, Di Stefano A, Turato G, et al. CD8+ T-lymphocytes in peripheral airways of smokers with chronic obstructive pulmonary disease. Am J Respir Crit Care Med. 1998;157(3 pt 1):822-826. [CrossRef]
 
Hodge G, Mukaro V, Holmes M, Reynolds PN, Hodge S. Enhanced cytotoxic function of natural killer and natural killer T-like cells associated with decreased CD94 (Kp43) in the chronic obstructive pulmonary disease airway. Respirology. 2013;18(2):369-376. [CrossRef]
 
Urbanowicz RA, Lamb JR, Todd I, Corne JM, Fairclough LC. Altered effector function of peripheral cytotoxic cells in COPD. Respir Res. 2009;10:53. [CrossRef]
 
Phillips B, Marshall ME, Brown S, Thompson JS. Effect of smoking on human natural killer cell activity. Cancer. 1985;56(12):2789-2792. [CrossRef]
 
Zeidel A, Beilin B, Yardeni I, Mayburd E, Smirnov G, Bessler H. Immune response in asymptomatic smokers. Acta Anaesthesiol Scand. 2002;46(8):959-964. [CrossRef]
 
Prieto A, Reyes E, Bernstein ED, 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(7):1578-1583. [CrossRef]
 
Spits H, Artis D, Colonna M, et al. Innate lymphoid cells—a proposal for uniform nomenclature. Nat Rev Immunol. 2013;13(2):145-149. [CrossRef]
 
Walker JA, Barlow JL, McKenzie AN. Innate lymphoid cells—how did we miss them? Nat Rev Immunol. 2013;13(2):75-87. [CrossRef]
 
Fuchs A, Vermi W, Lee JS, et al. Intraepithelial type 1 innate lymphoid cells are a unique subset of IL-12- and IL-15-responsive IFN-γ-producing cells. Immunity. 2013;38(4):769-781. [CrossRef]
 
Bernink JH, Peters CP, Munneke M, et al. Human type 1 innate lymphoid cells accumulate in inflamed mucosal tissues. Nat Immunol. 2013;14(3):221-229. [CrossRef]
 
Mjösberg JM, Trifari S, Crellin NK, et al. Human IL-25- and IL-33-responsive type 2 innate lymphoid cells are defined by expression of CRTH2 and CD161. Nat Immunol. 2011;12(11):1055-1062. [CrossRef]
 
Godfrey DI, MacDonald HR, Kronenberg M, Smyth MJ, Van Kaer L. NKT cells: what’s in a name? Nat Rev Immunol. 2004;4(3):231-237. [CrossRef]
 
Fairclough L, Urbanowicz RA, Corne J, Lamb JR. Killer cells in chronic obstructive pulmonary disease. Clin Sci (Lond). 2008;114(8):533-541. [CrossRef]
 
Kim CH, Johnston B, Butcher EC. Trafficking machinery of NKT cells: shared and differential chemokine receptor expression among V alpha 24(+)V beta 11(+) NKT cell subsets with distinct cytokine-producing capacity. Blood. 2002;100(1):11-16. [CrossRef]
 
Kelly-Rogers J, Madrigal-Estebas L, O’Connor T, Doherty DG. Activation-induced expression of CD56 by T cells is associated with a reprogramming of cytolytic activity and cytokine secretion profile in vitro. Hum Immunol. 2006;67(11):863-873. [CrossRef]
 
Urbanowicz RA, Lamb JR, Todd I, Corne JM, Fairclough LC. Enhanced effector function of cytotoxic cells in the induced sputum of COPD patients. Respir Res. 2010;11:76. [CrossRef]
 
Henkart PA, Sitkovsky MV. Cytotoxic lymphocytes. Two ways to kill target cells. Curr Biol. 1994;4(10):923-925. [CrossRef]
 
Kojima H, Shinohara N, Hanaoka S, et al. Two distinct pathways of specific killing revealed by perforin mutant cytotoxic T lymphocytes. Immunity. 1994;1(5):357-364. [CrossRef]
 
Chrysofakis G, Tzanakis N, Kyriakoy D, et al. Perforin expression and cytotoxic activity of sputum CD8+ lymphocytes in patients with COPD. Chest. 2004;125(1):71-76. [CrossRef]
 
Hodge S, Hodge G, Brozyna S, Jersmann H, Holmes M, Reynolds PN. Azithromycin increases phagocytosis of apoptotic bronchial epithelial cells by alveolar macrophages. Eur Respir J. 2006;28(3):486-495. [CrossRef]
 
Hogg JC, Chu F, Utokaparch S, et al. The nature of small-airway obstruction in chronic obstructive pulmonary disease. N Engl J Med. 2004;350(26):2645-2653. [CrossRef]
 
van der Strate BW, Postma DS, Brandsma CA, et al. Cigarette smoke-induced emphysema: a role for the B cell? Am J Respir Crit Care Med. 2006;173(7):751-758. [CrossRef]
 
Smyth LJ, Starkey C, Vestbo J, Singh D. CD4-regulatory cells in COPD patients. Chest. 2007;132(1):156-163. [CrossRef]
 

Figures

Figure Jump LinkFigure 1. A-F, Lymphocyte characterization in BAL and PB. Cells were stained with a multicolor test to characterize the lymphocyte cell types (A) CD8+ T cells, (B) CD4+ T cells, (C) the CD4/CD8 ratio, (D) natural killer (NK) cells, (E) natural killer T (NKT)-like cells, and (F) B cells, in NSs, Ss, CSs, and CEs. G-I, Representative flow cytometric dot plots of (G) CD4+ and CD8+ among CD3+ T cells, (H) NK cells, identified as CD3CD56+/CD16+ cells, and NKT-like cells, identified as CD3+CD56+/CD16+ cells, among CD45+ cells, and (I) B cells, identified as CD3CD19+ cells, among CD45+ cells. *P < .05; **P < .01; ***P < .001. CE = ex-smoker with COPD; CS = smoker with COPD; NS = never smoker; PB = peripheral blood; S = smoker.Grahic Jump Location
Figure Jump LinkFigure 2. A-D, Proportions of (A) CD8+ cells and (B) CD4+ cells, among CD16+ and/or CD56+ T cells (ie, NKT-like cells), and CD16+ and/or CD56+ cells among (C) CD8+ T cells and (D) CD4+ T cells in BAL. *P < .05; **P < .01; ***P < .001. See Figure 1 legend for expansion of abbreviations.Grahic Jump Location
Figure Jump LinkFigure 3. A-D, Characterization of T-cell differentiation in BAL and PB CD4+ cells using CD27 and CD45RA expression. (A) CM cells, (B) naive cells, (C) EM cells, and (D) effector cells, in NSs, Ss, and patients with COPD. E-F, Representative flow cytometric dot plots of CD27 stained together with CD45RA among CD3+CD4+ T cells in (E) BAL and (F) PB. **P < .01. CM = central memory; EM = effector memory. See Figure 1 legend for expansion of other abbreviations.Grahic Jump Location
Figure Jump LinkFigure 4. A-D, Expression of the activation marker CD69 among (A) CM, (B) naive, (C) EM, and (D) effector CD4+ T cells in BAL. *P < .05; **P < .01; ***P < .001. See Figure 1 and 3 legends for expansion of abbreviations.Grahic Jump Location
Figure Jump LinkFigure 5. Correlation between the percentage of CD8+ T cells in BAL from male smokers with COPD and the number of cigarettes smoked per day during the preceding 6 months. p[FDR] = P value corrected for multiple testing by means of false discovery rate according to Benjamini-Hochberg; rs = Spearman rank correlation coefficient.Grahic Jump Location

Tables

Table Graphic Jump Location
Table 1 —Characteristics and Lung Function Data of Never Smokers, Smokers, and Patients With COPD

Data are presented as median (range) unless indicated otherwise. CRQ = Chronic Respiratory Disease Questionnaire; Dlco = diffusion capacity of the lung for carbon monoxide; GOLD = Global Initiative for Chronic Obstructive Pulmonary Disease; NA = not applicable; RV = residual volume.

a 

P < .05 when compared with smokers.

b 

P < .001 when compared with never smokers.

c 

P < .001 when compared with smokers.

d 

P < .01 when compared with never smokers.

e 

P < .05 when compared with never smokers.

Table Graphic Jump Location
Table 2 —BAL Characteristics of Never Smokers, Smokers, Smokers With COPD, and Ex-Smokers With COPD

Data are presented as median (range) unless indicated otherwise.

a 

P < .001 when compared with never smokers.

b 

P < .01 when compared with smokers.

c 

P < .01 when compared with never smokers.

d 

P < .001 when compared with smokers.

e 

P < .001 when compared with smokers with COPD.

f 

P < .01 when compared with smokers with COPD.

g 

P < .05 when compared with smokers with COPD.

h 

P < .05 when compared with smokers.

References

Global strategy for the diagnosis, management, and prevention of COPD. GOLD website. http://www.goldcopd.org. Accessed November 25, 2013.
 
Löfdahl JM, Wahlström J, Sköld CM. Different inflammatory cell pattern and macrophage phenotype in chronic obstructive pulmonary disease patients, smokers and non-smokers. Clin Exp Immunol. 2006;145(3):428-437. [CrossRef]
 
Sköld CM, Hed J, Eklund A. Smoking cessation rapidly reduces cell recovery in bronchoalveolar lavage fluid, while alveolar macrophage fluorescence remains high. Chest. 1992;101(4):989-995. [CrossRef]
 
Willemse BW, ten Hacken NH, Rutgers B, Lesman-Leegte IG, Postma DS, Timens W. Effect of 1-year smoking cessation on airway inflammation in COPD and asymptomatic smokers. Eur Respir J. 2005;26(5):835-845. [CrossRef]
 
Miller M, Cho JY, Pham A, Friedman PJ, Ramsdell J, Broide DH. Persistent airway inflammation and emphysema progression on CT scan in ex-smokers observed for 4 years. Chest. 2011;139(6):1380-1387. [CrossRef]
 
Finkelstein R, Fraser RS, Ghezzo H, Cosio MG. Alveolar inflammation and its relation to emphysema in smokers. Am J Respir Crit Care Med. 1995;152(5 pt 1):1666-1672. [CrossRef]
 
Majo J, Ghezzo H, Cosio MG. Lymphocyte population and apoptosis in the lungs of smokers and their relation to emphysema. Eur Respir J. 2001;17(5):946-953. [CrossRef]
 
Calabrese F, Giacometti C, Beghe B, et al. Marked alveolar apoptosis/proliferation imbalance in end-stage emphysema. Respir Res. 2005;6:14. [CrossRef]
 
Saetta M, Baraldo S, Corbino L, et al. CD8+ve cells in the lungs of smokers with chronic obstructive pulmonary disease. Am J Respir Crit Care Med. 1999;160(2):711-717. [CrossRef]
 
O’Shaughnessy TC, Ansari TW, Barnes NC, Jeffery PK. Inflammation in bronchial biopsies of subjects with chronic bronchitis: inverse relationship of CD8+ T lymphocytes with FEV1. Am J Respir Crit Care Med. 1997;155(3):852-857. [CrossRef]
 
Turato G, Zuin R, Miniati M, et al. Airway inflammation in severe chronic obstructive pulmonary disease: relationship with lung function and radiologic emphysema. Am J Respir Crit Care Med. 2002;166(1):105-110. [CrossRef]
 
Hamann D, Baars PA, Rep MH, et al. Phenotypic and functional separation of memory and effector human CD8+ T cells. J Exp Med. 1997;186(9):1407-1418. [CrossRef]
 
Hamann D, Roos MT, van Lier RA. Faces and phases of human CD8 T-cell development. Immunol Today. 1999;20(4):177-180. [CrossRef]
 
Hamann D, Kostense S, Wolthers KC, et al. Evidence that human CD8+CD45RA+CD27- cells are induced by antigen and evolve through extensive rounds of division. Int Immunol. 1999;11(7):1027-1033. [CrossRef]
 
Okada R, Kondo T, Matsuki F, Takata H, Takiguchi M. Phenotypic classification of human CD4+ T cell subsets and their differentiation. Int Immunol. 2008;20(9):1189-1199. [CrossRef]
 
Vivier E, Tomasello E, Baratin M, Walzer T, Ugolini S. Functions of natural killer cells. Nat Immunol. 2008;9(5):503-510. [CrossRef]
 
Peralbo E, Alonso C, Solana R. Invariant NKT and NKT-like lymphocytes: Two different T cell subsets that are differentially affected by ageing. Exp Gerontol. 2007;42(8):703-708. [CrossRef]
 
Sandberg A, Sköld CM, Grunewald J, Eklund A, Wheelock AM. Assessing recent smoking status by measuring exhaled carbon monoxide levels. PLoS ONE. 2011;6(12):e28864. [CrossRef]
 
Mikko M, Forsslund H, Cui L, et al. Increased intraepithelial (CD103+) CD8+ T cells in the airways of smokers with and without chronic obstructive pulmonary disease. Immunobiology. 2013;218(2):225-231. [CrossRef]
 
Kohler M, Sandberg A, Kjellqvist S, et al. Gender differences in the bronchoalveolar lavage cell proteome of patients with chronic obstructive pulmonary disease. J Allergy Clin Immunol. 2013;131(3):743-751. [CrossRef]
 
Definition and classification of chronic bronchitis for clinical and epidemiological purposes. A report to the Medical Research Council by their Committee on the Aetiology of Chronic Bronchitis. Lancet. 1965;1(7389):775-779.
 
Rabe KF, Hurd S, Anzueto A, et al; Global Initiative for Chronic Obstructive Lung Disease. Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease: GOLD executive summary. Am J Respir Crit Care Med. 2007;176(6):532-555. [CrossRef]
 
Guyatt G. Measuring health status in chronic airflow limitation. Eur Respir J. 1988;1(6):560-564.
 
Wong DM, Varesio L. Depletion of macrophages from heterogeneous cell populations by the use of carbonyl iron. Methods Enzymol. 1984;108:307-313.
 
Benjamini Y, Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc, B. 1995;57(1):289-300.
 
Löfdahl JM, Cederlund K, Nathell L, Eklund A, Sköld CM. Bronchoalveolar lavage in COPD: fluid recovery correlates with the degree of emphysema. Eur Respir J. 2005;25(2):275-281. [CrossRef]
 
Röpcke S, Holz O, Lauer G, et al. Repeatability of and relationship between potential COPD biomarkers in bronchoalveolar lavage, bronchial biopsies, serum, and induced sputum. PLoS ONE. 2012;7(10):e46207. [CrossRef]
 
Barceló B, Pons J, Fuster A, et al. Intracellular cytokine profile of T lymphocytes in patients with chronic obstructive pulmonary disease. Clin Exp Immunol. 2006;145(3):474-479. [CrossRef]
 
Roos-Engstrand E, Ekstrand-Hammarström B, Pourazar J, Behndig AF, Bucht A, Blomberg A. Influence of smoking cessation on airway T lymphocyte subsets in COPD. COPD. 2009;6(2):112-120. [CrossRef]
 
Wright JL, Lawson LM, Pare PD, Wiggs BJ, Kennedy S, Hogg JC. Morphology of peripheral airways in current smokers and ex-smokers. Am Rev Respir Dis. 1983;127(4):474-477.
 
Wright JL, Hobson J, Wiggs BR, Pare PD, Hogg JC. Effect of cigarette smoking on structure of the small airways. Lung. 1987;165(2):91-100. [CrossRef]
 
Fletcher C, Peto R. The natural history of chronic airflow obstruction. BMJ. 1977;1(6077):1645-1648. [CrossRef]
 
Postma DS, de Vries K, Koëter GH, Sluiter HJ. Independent influence of reversibility of air-flow obstruction and nonspecific hyperreactivity on the long-term course of lung function in chronic air-flow obstruction. Am Rev Respir Dis. 1986;134(2):276-280.
 
Hodge G, Nairn J, Holmes M, Reynolds PN, Hodge S. Increased intracellular T helper 1 proinflammatory cytokine production in peripheral blood, bronchoalveolar lavage and intraepithelial T cells of COPD subjects. Clin Exp Immunol. 2007;150(1):22-29. [CrossRef]
 
Saetta M, Di Stefano A, Turato G, et al. CD8+ T-lymphocytes in peripheral airways of smokers with chronic obstructive pulmonary disease. Am J Respir Crit Care Med. 1998;157(3 pt 1):822-826. [CrossRef]
 
Hodge G, Mukaro V, Holmes M, Reynolds PN, Hodge S. Enhanced cytotoxic function of natural killer and natural killer T-like cells associated with decreased CD94 (Kp43) in the chronic obstructive pulmonary disease airway. Respirology. 2013;18(2):369-376. [CrossRef]
 
Urbanowicz RA, Lamb JR, Todd I, Corne JM, Fairclough LC. Altered effector function of peripheral cytotoxic cells in COPD. Respir Res. 2009;10:53. [CrossRef]
 
Phillips B, Marshall ME, Brown S, Thompson JS. Effect of smoking on human natural killer cell activity. Cancer. 1985;56(12):2789-2792. [CrossRef]
 
Zeidel A, Beilin B, Yardeni I, Mayburd E, Smirnov G, Bessler H. Immune response in asymptomatic smokers. Acta Anaesthesiol Scand. 2002;46(8):959-964. [CrossRef]
 
Prieto A, Reyes E, Bernstein ED, 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(7):1578-1583. [CrossRef]
 
Spits H, Artis D, Colonna M, et al. Innate lymphoid cells—a proposal for uniform nomenclature. Nat Rev Immunol. 2013;13(2):145-149. [CrossRef]
 
Walker JA, Barlow JL, McKenzie AN. Innate lymphoid cells—how did we miss them? Nat Rev Immunol. 2013;13(2):75-87. [CrossRef]
 
Fuchs A, Vermi W, Lee JS, et al. Intraepithelial type 1 innate lymphoid cells are a unique subset of IL-12- and IL-15-responsive IFN-γ-producing cells. Immunity. 2013;38(4):769-781. [CrossRef]
 
Bernink JH, Peters CP, Munneke M, et al. Human type 1 innate lymphoid cells accumulate in inflamed mucosal tissues. Nat Immunol. 2013;14(3):221-229. [CrossRef]
 
Mjösberg JM, Trifari S, Crellin NK, et al. Human IL-25- and IL-33-responsive type 2 innate lymphoid cells are defined by expression of CRTH2 and CD161. Nat Immunol. 2011;12(11):1055-1062. [CrossRef]
 
Godfrey DI, MacDonald HR, Kronenberg M, Smyth MJ, Van Kaer L. NKT cells: what’s in a name? Nat Rev Immunol. 2004;4(3):231-237. [CrossRef]
 
Fairclough L, Urbanowicz RA, Corne J, Lamb JR. Killer cells in chronic obstructive pulmonary disease. Clin Sci (Lond). 2008;114(8):533-541. [CrossRef]
 
Kim CH, Johnston B, Butcher EC. Trafficking machinery of NKT cells: shared and differential chemokine receptor expression among V alpha 24(+)V beta 11(+) NKT cell subsets with distinct cytokine-producing capacity. Blood. 2002;100(1):11-16. [CrossRef]
 
Kelly-Rogers J, Madrigal-Estebas L, O’Connor T, Doherty DG. Activation-induced expression of CD56 by T cells is associated with a reprogramming of cytolytic activity and cytokine secretion profile in vitro. Hum Immunol. 2006;67(11):863-873. [CrossRef]
 
Urbanowicz RA, Lamb JR, Todd I, Corne JM, Fairclough LC. Enhanced effector function of cytotoxic cells in the induced sputum of COPD patients. Respir Res. 2010;11:76. [CrossRef]
 
Henkart PA, Sitkovsky MV. Cytotoxic lymphocytes. Two ways to kill target cells. Curr Biol. 1994;4(10):923-925. [CrossRef]
 
Kojima H, Shinohara N, Hanaoka S, et al. Two distinct pathways of specific killing revealed by perforin mutant cytotoxic T lymphocytes. Immunity. 1994;1(5):357-364. [CrossRef]
 
Chrysofakis G, Tzanakis N, Kyriakoy D, et al. Perforin expression and cytotoxic activity of sputum CD8+ lymphocytes in patients with COPD. Chest. 2004;125(1):71-76. [CrossRef]
 
Hodge S, Hodge G, Brozyna S, Jersmann H, Holmes M, Reynolds PN. Azithromycin increases phagocytosis of apoptotic bronchial epithelial cells by alveolar macrophages. Eur Respir J. 2006;28(3):486-495. [CrossRef]
 
Hogg JC, Chu F, Utokaparch S, et al. The nature of small-airway obstruction in chronic obstructive pulmonary disease. N Engl J Med. 2004;350(26):2645-2653. [CrossRef]
 
van der Strate BW, Postma DS, Brandsma CA, et al. Cigarette smoke-induced emphysema: a role for the B cell? Am J Respir Crit Care Med. 2006;173(7):751-758. [CrossRef]
 
Smyth LJ, Starkey C, Vestbo J, Singh D. CD4-regulatory cells in COPD patients. Chest. 2007;132(1):156-163. [CrossRef]
 
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