Published ahead of print on January 18, 2008, doi:10.1165/rcmb.2007-0400OC
© 2008 American Thoracic Society DOI: 10.1165/rcmb.2007-0400OC Function-Associated Surface Molecules on Airway Dendritic Cells in Cigarette Smokers1 Department of Pneumology, University of Rostock, Rostock, Germany Correspondence and requests for reprints should be addressed to Marek Lommatzsch, M.D., Abteilung für Pneumologie, Klinik und Poliklinik für Innere Medizin, Universität Rostock, Ernst-Heydemann-Str. 6, 18057 Rostock, Germany. E-mail: marek.lommatzsch{at}med.uni-rostock.de
Airway dendritic cells (DCs) control pulmonary immune responses to inhaled particles. However, the profile of function-associated surface molecules on airway DCs in smokers is unknown. In this study, function-associated surface molecules were analyzed using four-color flow cytometry on myeloid DCs (mDCs) in bronchoalveolar lavage fluid (BALF) of cigarette smokers and never-smokers. Furthermore, the lung function was assessed directly before bronchoscopy in all participants. There was a 7-fold increase in total cell numbers in BALF of smokers, as compared with never-smokers. The percentage of mDCs among BALF cells and the expression of the maturation marker CD83 on mDCs did not differ between smokers and never-smokers. However, there was a strong increase in the expression of Langerin and CD1a (markers of Langerhans cells) on mDCs of smokers. Furthermore, mDCs of smokers were characterized by an increased expression of antigen presentation markers such as CD80 and CD86. By contrast, mDCs of smokers displayed a decreased expression of the lymph node homing receptor CCR7, as compared with mDCs of never-smokers. Decreased expression of CCR7 on mDCs, but not any of the other surface molecules studied, was specifically associated with airway obstruction and pulmonary hyperinflation in smokers. In conclusion, our data suggest that smoking affects the expression profile of function-associated surface molecules on airway mDCs. We provide the first evidence that a reduced CCR7 expression on airway mDCs is associated with airflow limitation in smokers.
Key Words: cigarette smoke • myeloid dendritic cells • bronchoalveolar lavage fluid • flow cytometry • chemokine receptors
Cigarette smoking is the main risk factor for the development of chronic obstructive pulmonary disease (COPD), the fourth most common cause of death worldwide (1, 2). The disease is associated with a specific inflammatory response around small airways, which contributes to the progression of the disease (3). The precise mechanisms leading to this type of inflammation are unknown. However, the presence of lymphocyte follicles in small airways of patients with COPD, and its association with disease severity, suggests that adaptive immune responses play a role in this disease (4). Airway dendritic cells (DCs) initiate and regulate adaptive immune responses in the lung (5). They form a highly sensitive sentinel network around the airways, and appear to be able to migrate through the intact epithelium to sample foreign antigens within the airway lumen (6). After antigen uptake, DCs migrate to the draining lymph nodes to present antigenic information to specialized lymphocytes, which organize an inflammatory response against the encountered antigen (5). The traffic of DCs is facilitated by a sequence of chemotactic stimuli, and the expression of corresponding chemokine receptors on DCs (7). DCs play a crucial role in the pathogenesis of airway inflammation in asthma (8, 9). However, there is limited information on the phenotype and the role of DCs in smokers and patients with COPD. It has been shown that smokers display increased amounts of Langerhans cells (a subtype of myeloid DCs) in the airways (10, 11). An immunohistochemical study revealed that Langerhans cells are increased in the airways of smokers with COPD (12). However, due to the methodological limitations of these studies, the relationship of Langerhans cells to the total group of myeloid DCs (mDCs), and the expression of function-associated surface molecules on mDCs in smokers, are still unknown. In addition, there is no information on the relationship between these surface molecules and the lung function of smokers. We have recently established a comprehensive flow cytometric method to systematically quantify and characterize DCs in human bronchoalveolar lavage fluid (BALF) (9, 13). This new method is the first to provide an accurate identification of the different DC subsets in human BALF (14). In addition, it offers the possibility to analyze a large panel of surface markers on airway DCs. Finally, by using untreated cells from human BALF, it avoids the risk of phenotypic changes that may occur during enzymatic treatment in studies using lung homogenates (9, 13). On the basis of this method, we have designed a study that analyzes, for the first time, function-associated surface molecules on BALF mDCs of smokers, and compares these findings with never-smokers and with the lung function of the participants.
Participants The participants were recruited using public notices in Rostock (Germany). Smokers were recruited using the following inclusion criteria: (1) age between 30 and 60 years, (2) smoking history of at least 10 pack-years, and (3) current smoking of at least 10 cigarettes per day. Never-smokers were recruited using the following inclusion criteria: (1) age between 30 and 60 years, (2) no history of smoking and no exposure to smoking partners or relatives at home. For both groups, exclusion criteria were as follows: (1) any history of chronic cardiac, pulmonary, or inflammatory diseases; (2) any regular oral or inhaled cardiac or pulmonary medication; (3) any signs of a respiratory tract infection within the last 2 weeks before bronchoscopy. The study was approved by the local ethics committee of Rostock (Germany). All participants gave their written informed consent.
Study Design
Bronchoalveolar Lavage and Flow Cytometry
Statistical Analysis Data were analyzed using SPSS (Chicago, IL). Parameters were normally distributed. Therefore, parameters were calculated as means (± SD) and correlation analyses were performed using the Pearsons correlation coefficient. The comparison of parameters between smokers and never-smokers was performed using the independent-samples t test. Probability values of P < 0.05 were regarded as significant.
Characteristics of the Participants Ten never-smokers and 40 current smokers were included in the study based on our inclusion and exclusion criteria. The characteristics of the participants, including smoking history, blood parameters, BALF parameters, and lung function are detailed in Table 2. There were no significant differences in age, sex distribution, and anthropometric parameters between the two groups. In peripheral blood, leukocyte counts and CRP levels were significantly increased in smokers (Table 2). The BALF of smokers was characterized by a more than 7-fold increase in total cell counts. Despite a significant difference in macrophage and lymphocyte percentages, the general distribution of leukocyte subsets in BALF did not differ between never-smokers and smokers (Table 2). Most lung function parameters were significantly worse in smokers than in never-smokers (Table 2).
Presence of mDCs in Blood and BALF The percentage of mDCs among leukocytes in peripheral blood was decreased in smokers, as compared with never-smokers (0.17 ± 0.07 versus 0.25 ± 0.09%, P < 0.01). However, total numbers of mDCs in peripheral blood did not differ between the groups (12,895 ± 5,456 versus 12,284 ± 5,328 cells/ml blood; P = 0.75). The percentages of mDCs among BALF cells did not differ between smokers and never-smokers (Table 3). Due to the strong increase in total BALF cell counts, the total number of mDCs in BALF was markedly increased in smokers (Table 3).
Characteristics of mDCs in BALF Among BALF mDCs of smokers, there was a strong increase in the percentage of Langerin-positive, CD1a-positive, and BDCA-1-positive DCs, as compared with never-smokers (Table 3). Strikingly, there was no overlap regarding the percentage of Langerin-positive cells among mDCs between smokers and never-smokers (Figure 3). The expression of Langerin correlated with the expression of CD1a (smokers: r = 0.68, P < 0.001; never-smokers: r = 0.78, P < 0.05), and, to a lesser extent, with BDCA-1 (smokers: r = 0.56, P < 0.001; never-smokers: r = 0.52, P = 0.15). The expression of BDCA-3 did not differ between the groups. BDCA-4 was expressed on mDCs in never-smokers, and this expression was stronger in smokers (Table 3). There was an increased expression of CD80, CD86, and mannose receptors, but a decreased expression of CCR5 and CCR7 on mDCs of smokers (Figure 2 and Table 3). In contrast, the expression of the maturation marker CD83 did not differ between the groups. However, the expression of CD83 correlated with the expression of CD86 on mDCs in both groups (smokers: r = 0.67, P < 0.001; never-smokers: r = 0.81, P < 0.01). The very low expression of DC-SIGN (< 3%) on mDCs precluded a reliable comparison between the groups (Figure 2).
DC Characteristics and Smoking History In the group of smokers, there was no correlation between the percentage of Langerin-positive cells among BALF mDCs and the number of pack-years (P = –0.16, r = 0.35) or the number of cigarettes smoked per day (P = –0.08, r = 0.62). In addition, there was no correlation between the total percentage of mDCs among BALF cells and the number of pack-years (P = –0.23, r = 0.15) or the number of cigarettes smoked per days (P = –0.18, r = 0.28). However, there was a significant decrease of CCR7 expression on mDCs with increasing numbers of pack-years (r = –0.36, P < 0.05) or cigarettes smoked per day (r = –0.31, P < 0.05) (Figure 4). The expression of other DC markers was not significantly correlated with the number of pack-years or the number of cigarettes smoked per day in the group of smokers (data not shown).
DC Characteristics and Lung Function The ratio of the forced expiratory volume in the first one second to the forced vital capacity (FEV1/FVC) in smokers was significantly correlated with the expression of CCR7 (but not CCR5) on mDCs (Figure 5). There were also correlations of CCR7-positive mDCs with other markers of airway obstruction, such as the peak expiratory flow (PEF; r = 0.36, P < 0.05) and the effective airway resistance (Reff; r = –0.39; P < 0.05). In addition, there was a significant negative correlation of CCR7-positive mDCs with hyperinflation of the lungs (RV/TLC: ratio of the residual volume to the total lung capacity) (Figure 5). The association between the FEV1 (% predicted) and CCR7-positive mDCs did not reach statistical significance (r = 0.29, P = 0.08). Neither the percentages or total cell counts of DCs, nor any other of the DC markers, displayed a consistent association with lung function parameters in smokers (data not shown).
This study is the first to identify the expression profile of function-associated surface molecules on airway mDCs in smokers. In addition, an association was found between the expression of CCR7 on mDCs and airflow limitation in smokers. Thus, the current study provides important new insights into the nature of airway mDCs in smokers, and its relationship to lung function. Despite a large literature on the role of airway DCs in asthma (8, 9, 15, 16), there is limited information on airway DCs in smokers. It has been shown in animal models that there is an increase in airway mDCs (17) and Langerhans cells (18) after smoke inhalation. Human studies described increased numbers of Langerhans cells in BALF (10) and in lung parenchyma (11) of smokers. Recently, an immunohistochemical study demonstrated an increase in Langerhans cells in the airways of smokers with COPD, as compared with smokers without COPD and nonsmokers (12). Our study is the first to quantify airway Langerhans cells in relation to the total population of airway mDCs in smokers. We show that the percentage of Langerin-positive and CD1a-positive cells among mDCs, but not the total percentage of mDCs, is strongly enhanced in the airways of smokers. Both Langerin (CD207) and CD1a have been described as markers of pulmonary Langerhans cells (19). As a proof of concept, we found a strong correlation between these markers on airway mDCs. Langerin appears to be the most specific marker of Langerhans cells, since activation of this surface molecule induces the formation of Birbeck granules, the defining element of Langerhans cells (20). It is of note that there was no overlap regarding the percentage of Langerin-positive cells among mDCs between smokers (more than 50% of all mDCs) and never-smokers (less than 50% of all mDCs). This clear-cut difference suggests that an increase in the Langerhans cell phenotype is a specific feature of airway mDCs in smokers. Beyond the Langerhans cell phenotype, mDCs of smokers displayed an increased expression of the antigen presentation markers CD80 and CD86 (21). In contrast, there was a decreased expression of the chemokine receptor CCR5, a marker for the attraction of immature DCs to the lung, and CCR7, a marker for the attraction of mature DCs to the lymph nodes (7). The profile of CD80, CD86, and CCR5 expression would be compatible with a more mature DC phenotype, whereas the decrease of CCR7 would be compatible with a more immature DC phenotype. Due to the multiple functions of mannose receptors in the innate and adaptive immune system, the observed increase in mannose receptors may relate to both immature and mature DCs (22). Thus, our observations do not clearly point to a more mature or immature phenotype of DCs in smokers. This is supported by the finding that the expression of the maturation marker CD83 did not differ between the groups. However, the phenotype of mDCs may be a direct result of the cigarette smoke itself. Cigarette smoke extracts (but not nicotine alone) suppress the maturation-associated expression of CCR7 on human mDCs in vitro (23). This is in line with our in vivo observation that CCR7 expression on mDCs decreased with increasing smoke exposure of the participants. Therefore, given the essential role of CCR7 for the migration of mDCs to draining lymph nodes (24, 25), cigarette smoke could reduce the migratory potential of mDCs. On the other hand, uptake of cigarette smoke particles may explain the up-regulation of antigen-presenting molecules, and the down-regulation of the immaturity marker CCR5. Thus, cigarette smoke might have a paradoxical effect on airway mDCs in smokers: stimulating DC maturation and antigen presentation, but suppressing DC migration to the draining lymph nodes. It has recently been postulated that the accumulation of DCs in the airways of smokers with COPD is due to an enhanced local expression of DC attracting chemokines such as CCL20 (12). Our data suggest that the accumulation of airway DCs in smokers may also be due to a decreased homing of DCs to the draining lymph nodes. The total percentage of mDCs or Langerhans cells in BALF did not show a significant association with the lung function of the smokers. In contrast, there was a consistent and specific association between reduced CCR7 expression on mDCs, and airflow limitation and pulmonary hyperinflation in smokers. The precise mechanisms linking reduced CCR7 expression on mDCs and airway obstruction remain to be elucidated. It might, however, be speculated that an impaired homing of mDCs to the lymph nodes results in an accumulation of mDCs in the airways. This accumulation could stimulate local adaptive immune responses which trigger airway remodeling and obstruction. It is of note that in COPD, a disease primarily related to cigarette smoking, the severity of airway obstruction is strongly correlated with the percentage of airways containing lymphoid follicles (3). It has, therefore, been postulated that excessive local adaptive immune responses are key elements in the pathogenesis of COPD (4). Our data suggest that cigarette smoke may stimulate these local immune responses by impairing the homing of airway DCs to the lymph nodes, thus promoting local antigen presentation within the airway wall. Any reference to COPD, however, needs to be done with caution, since patients in our study were selected based on the smoking history, and not on the presence of COPD symptoms. Although this study clearly points out to a relationship between smoking and reduced DC CCR7 expression, the relationship between CCR7 and airflow limitation or hyperinflation is not robust and further studies are necessary to define that relationship using a cohort of patients with well-defined COPD. In conclusion, these data indicate that smoking affects the expression profile of function-associated surface molecules on airway mDCs. We provide first evidence that a reduced CCR7 expression on airway mDCs might be associated with airflow limitation in smokers.
The authors thank Petra Thamm, Jana Brandt, Gesine Fastnacht, and Christiane Beil for excellent technical assistance.
This work was supported by Deutsche Forschungsgemeinschaft (DFG)(Grant LO 1145/2-1). 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: 10.1165/rcmb.2007-0400OC on January 18, 2008 Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Received in original form November 6, 2007 Accepted in final form January 8, 2008
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