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Published ahead of print on December 23, 2004, doi:10.1165/rcmb.2004-0196OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 32, pp. 262-267, 2005
© 2005 American Thoracic Society
DOI: 10.1165/rcmb.2004-0196OC

Primary Human Alveolar Type II Epithelial Cell CCL20 (Macrophage Inflammatory Protein-3{alpha})–Induced Dendritic Cell Migration

Andrew J. Thorley, Peter Goldstraw, Alan Young and Teresa D. Tetley

Lung Cell Biology, National Heart and Lung Institute, Imperial College; Department of Thoracic Surgery, Royal Brompton and Harefield NHS Trust, London; and AstraZeneca R&D, Loughborough, United Kingdom

Correspondence and requests for reprints should be addressed to T. D. Tetley, Lung Cell Biology, National Heart & Lung Institute, Dovehouse Street, London SW3 6LY, UK. E-mail: t.tetley{at}imperial.ac.uk


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Inhalation of antigenic matter stimulates rapid recruitment of dendritic cells (DCs) into the lung. Recent studies propose that the chemokine CCL20 (macrophage inflammatory protein-3{alpha}) may play an important role in DC recruitment. We previously showed that primary human alveolar type II epithelial (ATII) cells are a rich source of chemokines and so hypothesized that the ATII cell produces CCL20 and might therefore be a key regulator of DC recruitment into the lung. Here, we show that primary human ATII cells, but not human alveolar macrophages, produce CCL20 both constitutively (403.5 ± 85.4 pg/ml; 24 h) and in response to endotoxin (lipopolysaccharide) exposure (1,525.0 ± 169.4 pg/ml; 1 µg/ml lipopolysaccharide; 24 h) in a time- and dose-dependent manner. In addition, we show that peripheral blood monocyte-derived CD1a+ DCs migrate in response to conditioned media from ATII cells but not those from alveolar macrophages; DC migration was significantly correlated with the amount of CCL20 (r2 > 0.9; P < 0.05) detected in the media but not with any other chemokine measured. We therefore conclude that the alveolar epithelium is an important source of CCL20 in the lung and that the ATII cell may play a critical role in controlling the movement of DCs through the lung both under normal and inflammatory conditions.

Key Words: dendritic cell • alveolar epithelium • chemokine


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
The lung is exposed to a large number of inhaled pathogens and other organic matter each day and, as such, an efficient and rapid immunologic response is essential. One of the most important effector cells in the immune system is the dendritic cell (DC). DCs are professional antigen-presenting cells derived from the bone marrow and are found in nearly every tissue of the body. They are the sentinels of the immune system and play an essential and unique role in initiating and regulating primary immune responses to invading pathogens (1). A major function is to sample and process pathogens, and thus to prime naive T cells, either locally or within the draining lymph nodes (2–4).

A significant body of work highlights the role of DC in asthma. An early event in atopic asthma is presentation of antigen by DC to T cells (3, 4) and increased Th2 cell numbers. Rapid DC recruitment after allergen challenge involves DC receptor switching and altered chemokine production and profile by resident lung cells (5). The DC2 subset may also be responsible for orchestrating the Th2 responses in individuals with allergic rhinitis and asthma (6–8). There are a number of studies investigating the role of DCs in smoking-related diseases, such as histiocytosis X, most commonly seen in young male cigarette smokers, but relatively less is understood about the exact role of DCs in COPD. A direct link between cigarette smoke exposure and DC numbers has been shown using histopathology, where increased Langerhans cells were detected in the alveolar parenchyma, but not in the bronchioles. The Langerhans cells were found closely associated with areas of alveolar type II pneumocyte (ATII) hyperplasia (9). However, a recent investigation of DCs in cigarette smoke–exposed mice that develop emphysema shows a reduction in lung DC numbers (10). This suggests that DC recruitment fluctuates after cigarette smoke exposure, possibly reflecting different stages of the disease process.

The exact processes of DC recruitment to the lung are still being unraveled. The chemokine, CCL20 (also termed MIP-3{alpha}, LARC, and Exodus-1), has been shown to be important in CD1a+ DC (Langerhans precursor cell) migration in vitro and is highly expressed in inflamed epithelium in the skin and tonsil crypts (11). CCL20 is expressed and released by primary human bronchial epithelial cells (12) in response to cytokines and respirable particulate matter. Release of CCL20 by pulmonary epithelium may therefore be a crucial, early event in DC, and particularly Langerhans cell, trafficking into the lung.

Previous in vitro studies in this laboratory show that primary human ATII cells are a rich source of chemokines (13). We hypothesized that ATII cells and macrophages may be responsible, in part, for DC migration in the peripheral lung parenchyma by releasing CCL20. We isolated primary human ATII cells and macrophages using established methods (13, 14) to investigate the effect of lipopolysaccharide (LPS) on release of a panel of chemokines, in particular CCL20, and compared the effect of chemokines released by macrophages and ATII cells on monocyte-derived DC migration. LPS-stimulated human ATII cells produced high levels of CCL20; macrophages did not. Migration of monocyte-derived CD1a+ DC was dependent upon the amount of CCL20 in conditioned media from ATII cells, suggesting an important role for ATII cell–derived CCL20 in alveolar immune response mechanisms.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Isolation of Primary Human Type II Epithelial Cells and Alveolar Macrophages
ATII cells and macrophages were isolated from lung of grossly normal appearance after resection for lung carcinoma, with the approval of the Royal Brompton and Harefield Ethical Committee, as previously described (n = 9 consecutive subject samples [14]). Briefly, lung sections were perfused by injection of 0.9% sterile sodium chloride (saline) until the draining lavage ran clear and the cell count was less than 1 x 104 cells/ml. The draining saline lavage was collected and centrifuged at 1,300 rpm (290 x g) for 10 min at 20°C. The cell pellet was re-suspended in serum-free low protein hybridoma medium (LPHM) (Invitrogen, Paisley, UK) containing 1% penicillin/streptomycin/glutamine (PSG; Invitrogen) and plated in 24-well tissue culture plates (VWR; 0.5 x 106 macrophages/well). After 3 h macrophages had adhered and the media was removed and the wells were washed to remove nonadherent cells. The macrophages were maintained in serum-free LPHM + 1% PSG.

To isolate epithelial ATII cells, tissue was perfused and inflated with trypsin (0.25% in HBSS; Sigma, Poole, UK) and incubated at 37°C for 45 min; trypsin was replaced twice during this time. The tissue was finely chopped in the presence of newborn calf serum (NCS; Invitrogen). The chopped tissue was then incubated with DNase (250 µg/ml; Sigma) and the mixture passed through a 300-µm filter, followed by a 40-µm filter to remove large tissue debris. The cell suspension was then centrifuged at 1,300 rpm (290 x g) for 10 min and the resulting pellet re-suspended in DCCM-1 media (React Scientific, Troon, UK) containing 50 µg/ml DNase. These cells were incubated in tissue culture flasks for 2 h at 37°C in a humidified incubator to allow differential adherence of mononuclear cells, which were subsequently stained for the mononuclear cell markers described below.

After 2 h the nonadherent ATII cells were removed and the cell suspension centrifuged at 1,300 rpm (290 x g) for 10 min. The cell pellet was then resuspended in DCCM-1 containing 10% NCS and 1% PSG at a concentration of 1 x 106 cells/ml. Cells were then seeded at 1 x 106 ATII cells per well. Cells reached confluence by 48 h.

Dendritic Cell Culture
Blood monocyte preparation. One hundred milliliters of venous blood were collected on three separate occasions from three healthy volunteers, and the erythrocytes were removed using dextran sedimentation. The resulting leukocyte-rich plasma was layered onto 2.5-ml Nycoprep 1.068 (Robbins Scientific, Shirley, UK) and centrifuged (600 x g, 15 min, 20°C). The plasma and cells were then collected into separate tubes and centrifuged (700 x g, 10 min, 20°C). The monocyte pellet was then resuspended in platelet-poor plasma (5% plasma/95% saline) and centrifuged again (78 x g, 10 min, 20°C) to remove platelets. This was repeated four times.

The resulting cells were resuspended in DCCM-1 containing 1% PSG and 10% FCS at 1 x 106/ml and plated in 6-well tissue culture plates at 2 x 106 per well. After 1–1.5 h the wells were washed to remove nonadherent cells. Following this, cells were > 95% pure with a typical yield of 15–20 x 106 cells/100 ml blood.

Dendritic cell differentiation. Monocytes were maintained in complete media containing 50 ng/ml GM-CSF, 10 ng/ml transforming growth factor-ß1, and 1,000 U/ml interleukin (IL)-4 at 37°C (R&D Systems, Abingdon, UK) for 7 d (15, 16). One milliliter of fresh media containing these cytokines was added at days 3 and 5 without removing the old media. The resulting immature DCs exist in suspension and can be easily removed and used in migration assays. Cytospin preparations were air-dried and immunostained for CD1a, as described below.

LPS Stimulation of ATII Cells and Alveolar Macrophages
Cells from three patient tissue samples were prepared as described above. ATII cells and macrophages were then cultured in serum-free LPHM for the 24 h before addition of LPS (Escherichia coli 055:B5; Sigma). The media were then removed and the cells were incubated with LPS at concentrations of 1, 10, 100, or 1,000 ng/ml, in serum-free LPHM. Each condition was performed in triplicate. The resulting conditioned media were aspirated and the secreted chemokines measured by ELISA.

Conditioned media were also generated from LPS-stimulated ATII epithelial cells or macrophages, which were stimulated with LPS as described above, to use in the DC migration assay. After stimulation for 24 h with 1,000 ng/ml LPS, the medium was removed and the cells washed thoroughly to remove any residual LPS. Fresh serum-free medium was then added and the cells cultured for a further 24 h. The LPS-free conditioned media from this 24-h time period were then used for DC migration assays.

Measurement of Chemokines by ELISA
DuoSet (R&D Systems) kits were used to measure chemokines released from ATII cells and alveolar macrophages after LPS stimulation. Briefly, 96-well Maxisorp plates (VWR) were incubated overnight with the relevant primary mouse anti-human antibody. After washing and blocking of the plate the following day, duplicate aliquots of conditioned media were added and incubated for 2 h at room temperature, and the wells were then washed. A secondary biotinylated goat anti-human antibody was added at room temperature for a further 2 h, then washed. Streptavidin conjugated to horseradish peroxidase (HRP) was added for 20 min and after washing, bound conjugate was detected through the addition of HRP substrate containing hydrogen peroxide and tetramethylbenzidine to produce a yellow color. After the addition of a stop solution (2N H2SO4) the absorbance was immediately determined at 450 nm using a spectrophotometer. Each ELISA assay plate contained a paired set of standards.

Immunocytochemistry
Adherent mononuclear cells derived from the lavaged tissue and trypsinised, chopped lung sections were stained for CD1a and CD68, whereas ATII cell monolayers were stained for CCL20. Cells were prepared and cultured as described above. The medium was removed and the cells washed in HBSS (Sigma). Cells were then left to air dry before being fixed in acetone:methanol 1:1 (Sigma). In addition, monocyte-derived DC cells were stained for CD1a. Dako Envision kits (Dako, Ely, UK) were used for immunostaining. Briefly, a peroxidase block was used to inhibit any cellular peroxidase activity that may interfere with staining. The primary mouse anti-human monoclonal antibody was then added to the wells and the plates incubated for 40 min. After this the wells were washed and secondary antibody added. The plate was then incubated for a further 40 min, secondary antibody removed, and the cells were washed again before adding the chromogenic DAB solution. The substrate solution was left for 10 min and then washed off with water. Cells were counterstained using Mayer's haematoxylin (Sigma) and then washed with 37 mM ammonia (VWR) as a bluing reagent.

Dendritic Cell Migration Assay
Blood monocyte–derived immature CD1a+ DCs were aspirated from culture plates, centrifuged and resuspended in LPHM media at a concentration of 1 x 106/ml. One hundred microliters of the cell suspension were then placed into the upper chamber of a Transwell insert (8 µm pore membrane; Corning BV, Schiphol-Rijk, The Netherlands). Three hundred microliters of LPHM containing recombinant CCL20 (R&D Systems) or macrophage- or ATII cell–conditioned medium (n = 3 subject samples) were placed into companion 24-well plates (i.e., in the lower chamber). Plates were then incubated for up to 24 h in a humidified incubator. After incubation the cell culture insert was removed and the nonmigratory cells in the upper chamber were aspirated and wiped away from the membrane. The underside of the membrane was then examined for adherent migratory cells; this was always negative. The media remaining in the tissue culture wells were aspirated and examined microscopically for nonadherent migratory cells, but were always negative. Remaining, adherent, migratory DC cells in the lower chamber were covered with HBSS and counted under a light microscope. Migration was expressed as cells per field of vision, n = 5 fields/well. The possible effect of chemokinesis (i.e., nonspecific migration) was determined by preincubating DC with conditioned medium from patient C in a tissue culture insert for 5 min, before addition of the same conditioned media to the lower chamber and processing to determine DC migration in the same way as the test samples. The effect of supermaximal levels of blocking mouse monoclonal antibodies to CCL20 (25 µg/ml), CXCL1 (Gro{alpha}; 50 µg/ml), CXCL8 (IL-8; 25 µg/ml), CCL5 (RANTES; 5 µg/ml), CCL2 (MCP-1; 10 µg/ml), and CCL3 (MIP-1{alpha}; 25 µg/ml) (all from R&D Systems) was determined by addition of antibodies to conditioned media in the lower chamber of a parallel set of experiments. The supermaximal antibody levels that were used were based on the concentration of chemokine in the conditioned media (CCL20, 6,271 ± 126 pg/ml; CXCL1, 33,375 ± 236 pg/ml; CXCL8, 27,168 ± 198 pg/ml; CCL5, 55 ± 9 pg/ml; CCL2, 9,258 ± 78 pg/ml; CCL3, 313 ± 43 pg/ml), as recommended by the manufacturer, to completely block chemokine activity. As the appropriate negative controls, the effect of addition of the relevant IgG isotypes (IgG1 and IgG2A; Dako) to the conditioned media was also investigated. The specificity of DC migration toward CCL20 was further determined by addition of increasing concentrations of blocking antibody to CCL20 to a constant amount (6,000 µg/ml) of CCL20 in the lower chamber. Antibodies were free of added preservatives or carrier proteins.

Statistical Analyses
Data are presented as mean ± SE. A one-way ANOVA was used to analyze the time- and dose-dependent effects of LPS on CCL20 release from ATII cells and dose-dependent effects of conditioned media on DC migration. Paired t tests were used to determine significant differences due to LPS treatment and responses to CCL20 and conditioned media. A P value < 0.05 was considered to be statistically significant.


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Immunostaining of Mononuclear Cell Populations
Two distinct populations of mononuclear cells were isolated and identified from the resected lung tissue. Using immunocytochemistry, it was shown that the adherent mononuclear cells isolated by saline lavage of the lung tissue contained no CD1a+ cells and were CD68+ CD1a– (CD68+, mean 96.2 ± 1.1%; n = 5, ~ 200 cells/field) and therefore macrophages (Figure 1), whereas adherent mononuclear cells subsequently isolated from the trypsinised tissue contained no CD68+ cells and contained a high proportion of CD68– CD1a+ (CD1a+ 90 ± 1.4%; n = 6, ~ 100 cells/field; Figure 1), indicating mostly a Langerhans cell phenotype.



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  		Figure 1. Immunostaining of mononuclear cells obtained from saline lavage of lung tissue and trypsinized lung tissue (A) and ATII epithelial cell monolayers (B). The mononuclear cells isolated by saline lavage are CD68+CD1a–, indicating an alveolar macrophage phenotype, whereas those obtained from trypsinized minced tissue are CD68–CD1a+, indicating a Langerhans precursor dendritic cell lineage. CCL20 was expressed by resting ATII cells (ii) and after 1 µg/ml LPS stimulation (iii). (i) Negative antibody isotype control.

 
CCL20 Production by Primary Human Type II Epithelial Cells and Macrophages
To assess whether primary human ATII cells produced CCL20, we investigated the release of the chemokine in freshly isolated cells, confluent untreated ATII cell monolayers, and after stimulation with LPS. Immunocytochemistry (Figure 1B) and ELISA (Figure 2) showed that freshly isolated ATII cells and ATII cell monolayers produced CCL20 under resting conditions and in response to LPS. Release after LPS exposure was found to be dose- and time-dependent and did not plateau over 24 h (Figure 2). The same experiments were performed using primary human alveolar macrophages. However, macrophages did not produce CCL20 either resting or in response to LPS (data not shown; n = 6 subject samples).



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  		Figure 2. Time- and dose-dependent release of CCL20 from ATII cells after LPS stimulation. Data expressed as mean ± SE (n = 6 patients). ***Migration is significantly increased above equivalent time untreated control (P < 0.0001).

 
In Vitro Migration of DCs Derived from Peripheral Blood Monocytes
To investigate whether blood monocyte–derived CD1a+ DCs migrated in response to CCL20, a standard invasion chamber technique was used. Using this system we showed that DCs derived from peripheral blood monocytes migrated in response to recombinant human CCL20 in a dose-dependent manner. Co-incubating the highest concentration of recombinant CCL20 with blocking antibodies to CCL20 significantly inhibited migration by ~ 80% (P < 0.0001; Figure 3A). In addition, when a constant amount of recombinant CCL20 was incubated with increasing concentrations of blocking antibody, DC migration was inhibited in a dose-dependent fashion, plateauing at ~ 70% inhibition (Figure 3B). This confirmed that DC migration was specific and in response to the active CCL20 in the lower chamber.



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  		Figure 3. (A) Dose-dependent migration of peripheral blood monocyte derived CD1a+ DC in response to human recombinant CCL20 (pg/ml) and blockade by monoclonal CCL20 antibody (Ab; 25 µg/ml). (B) Inhibitory effect of increasing amounts of CCL20 blocking antibody on CD1a+ DC migration toward 6,000 pg/ml CCL20. Data expressed as the mean ± SE (n = 3 separate samples from healthy volunteer subjects). *** Migration toward CCL20 is significantly greater than control (P < 0.0001); ### Migration toward CCL20 + Ab is significantly less than migration toward 6,000 pg/ml CCL20 alone (P < 0.0001).

 
After validation of the assay with recombinant CCL20, DC migration toward conditioned media from LPS-stimulated alveolar macrophages and ATII cells was examined. The conditioned media from ATII cells from three separate patients caused a significant increase in migration (P < 0.0001; Figure 4). Migration in response to conditioned media was significantly correlated with the amount of CCL20 (r2 > 0.9; P < 0.05) in the conditioned media. Migration did not correlate with any other chemokines measured in the media (CCL2, –0.98; CXCL1, 0.5; CCL5, –0.2; CCL3, –0.15; and CXCL8, –0.1; P > 0.05). In particular, there was no significant increase in migration in response to conditioned media from alveolar macrophages (Figure 4), even though alveolar macrophages produced all the other chemokines measured in ATII cell conditioned media, with the exception of CCL20. Preincubation of DC in conditioned media to measure nonspecific migration (possibly chemokinesis) showed that this might account for a small proportion (< 10%) of the total migration toward ATII cell conditioned media from Subject C (Figure 4).



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  		Figure 4. Effect of conditioned media from LPS-stimulated ATII cells from three patients (TII A, TII B, and TII C) and conditioned media from one representative alveolar macrophage preparation (Macrophage) on peripheral blood monocyte–derived CD1a+ DC migration. Effect of dual stimulation of CD1a+ DC migration is also shown as conditioned media from Patient C with 1 µg/ml LPS added (TII C + LPS; control = media + 1 µg/ml LPS). Migration after preincubation of DC in the same conditioned media illustrates nonspecific migration (Pre-Incubated). Levels of CCL20 in conditioned media are shown along the x-axis. Data expressed as the mean ± SE (n = 3 patients). *** Migration toward media control was significantly less than migration (i) toward ATII cell–conditioned media (P < 0.001), (ii) toward media + LPS, and (iii) by preincubated cells (P < 0.0006). ns, no significant difference between treatments.

 
The possible effect of dual signals on migration was also investigated by examining the effect of both LPS and conditioned media from ATII cells. LPS alone caused a smaller yet significant increase in migration (P < 0.0006). Addition of LPS to the conditioned media from subject C ATII cells had a small but nonsignificant additive effect on DC migration (Figure 4).

For all subsequent migration studies, the ATII cell conditioned media that caused the greatest migration was used (ATII C; Figure 4).

To further assess the relative contribution of each of the known chemokines to DC migration, supermaximal concentrations of blocking antibodies to each chemokine were added separately, based on the concentration of each chemokine in the media and the manufacturer's recommendations. The effect of IgG1 and IgG2A did not differ from each other and had only a small inhibitory effect on DC migration (IgG1 shown in Figure 5). Antibody to CCL20 was most effective, being significantly more inhibitory than any of the other chemokine-blocking antibodies (P < 0.0001; Figure 5). Nevertheless, the blocking antibodies to CCL2 and CCL5 also caused a significant (P < 0.0001), but lower, degree of inhibition of DC migration (15–30%), which was above that of the IgG control. Incubation of conditioned media with all the antibodies combined, at the same concentration as that used alone, caused a slightly greater (~ 8%) degree of inhibition than that of CCL20 antibody alone (P < 0.05), supporting a possible small contribution of CCL2 and CCL5 to DC migration. Incubation with antibodies against all the chemokines with the exception of CCL20 caused only a small inhibition of migration, which interestingly did not reach that of CCL2- and CCL5-blocking antibodies when they were used alone (Figure 5).



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  		Figure 5. The inhibitory effect on DC migration of addition of specific chemokine blocking antibodies to ATII cell–conditioned media. Shown as percent inhibition of DC migration toward conditioned medium from subject C (TII C) by supermaximal concentrations of blocking monoclonal antibodies to CCL3, CXCL1, CXCL8, CCL2, CCL5, and CCL20 used alone and combined (All Abs), or with all the antibodies excluding CCL20 antibody (All Abs Excl CCL20). Data expressed as the mean ± SE (n = 3 separate monocyte-derived DC samples from healthy volunteer subjects). *** There is significantly greater inhibition of DC migration when specific chemokine blocking antibodies are added to conditioned media compared with IgG control (P < 0.0001). ### There is significantly greater inhibition of DC migration when blocking antibody to CCL20 is added compared with addition of blocking antibodies to each of the other chemokines (P < 0.0001). # Inhibition of migration by all blocking antibodies combined is significantly greater than for blocking antibody to CCL20 Ab alone (P < 0.05). {dagger}{dagger}{dagger} Incubation with all antibodies but excluding CCL20 Ab (All Abs Excl CCL20) resulted in significantly less inhibition of migration compared with incubation with all the antibodies (All Abs) (P < 0.0001).

 

   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
This study shows that human ATII epithelial cells release CCL20 constitutively and in a dose- and time-dependent manner in response to LPS; in contrast, human alveolar macrophages obtained from the same subject samples did not release CCL20 under the same culture conditions. Furthermore, migration of immature CD1a+, Langerhans precursor cells toward conditioned media from ATII cells was related to, and was almost entirely dependent upon, CCL20 concentration. Pulmonary DCs rapidly increase in response to bacterial infection (17), allergen (18, 19) and other exogenous agents (20). A recent study showing that bronchial epithelial cell–derived CCL20 may be crucial in this process in the large airways (12, 21) and the current study of respiratory epithelium, showing stimulated release of CCL20 by LPS-treated ATII cells, suggest a major role for epithelium-derived CCL20. Because CCL20 is a particularly powerful chemokine for Langerhans cells (11, 22), exerting its chemotactic effects via the CCR6 receptor (23), the current study describes a mechanism of selective and specific recruitment of Langerhans cells to the pulmonary epithelium and an important immunoregulatory role for ATII cells in the respiratory unit.

Freshly isolated and unstimulated ATII cells in vitro released significant levels of CCL20 over 24 h, and immunostaining of unstimulated, adherent monolayers was also positive. This suggests constitutive synthesis and release of this chemokine by alveolar epithelial cells. Alternatively, it is possible that the lung tissue samples used for this study were inflamed, or that proteolytic release of epithelial cells stimulated basal CCL20, resulting in basal release in vitro that may not normally exist in vivo. However, studies by others invariably show constitutive expression of CCL20 mRNA by a variety of human tissues, including normal adult and fetal lung (23, 24). Constitutive expression of low levels of CCL20 by mucosal tissues such as the pulmonary epithelium would help to maintain the mucosal homeostasis by ensuring constant recruitment of inflammatory cells in the face of continuous antigen challenge (24). Furthermore, CCL20 expression by the epidermis of the skin in vivo (24, 25), and by blood monocytes and large airway bronchial epithelium in vitro (12, 26), is upregulated by cytokines such as IL-1, TNF-{alpha}, and IFN-{gamma}, supporting a role for increased CCL20 in recruiting immunomodulatory cells during inflammation.

We were surprised that LPS-stimulated macrophages neither released CCL20 nor generated conditioned media that stimulated CD1a+ DC recruitment, suggesting that they do not directly orchestrate CD1a+ DC recruitment. Power and colleagues (23) showed CCL20 mRNA expression by macrophages, and showed that LPS-stimulated peripheral blood monocytes release CCL20 at low levels (24). We therefore hypothesized that LPS-stimulated macrophages might also release CCL20. It is possible that LPS is the wrong stimulus to trigger macrophage CCL20 secretion. Alternatively, macrophages may exert control over pulmonary DC recruitment indirectly, by releasing IL-1, TNF-{alpha}, and other cytokines that are known to stimulate epithelial cells to release CCL20 (12, 24–26). This possibility is currently being investigated in this laboratory. Furthermore, macrophages may release other factors that might be important in recruiting other DC subsets, but this was not examined in this investigation.

For the DC migration studies, we used DC that were derived using a method that stimulates monocytes to develop into precursors of Langerhans cells (i.e., media containing TGF-ß as well as GM-CSF and TNF-{alpha} [15, 16]). DC derived using this technique have been extensively characterized and are enriched with CD1a+, Langerin-immunopositive cells that contain Birkbeck granules by electron microscopy, all features of Langerhans cells. We wanted to study Langerhans cells because these cells have been described to be closely associated with ATII epithelial cell hyperplasia in human lung tissue sections (9). In addition, we discovered that when using a method specifically designed to isolate ATII cells from whole lung tissue we also liberated CD1a+ mononuclear DC, suggesting specific homing of these cells to the alveolar epithelium and an important functional role at the air interface of the alveolar unit.

A number of different subsets of DC exist in the lung, each distinguished by a different pattern of cell surface marker expression (3, 4), but they are not yet fully characterized, particularly in humans. Much of our knowledge of DC biology stems from studies of skin and from studies of animal models. DC migration is tightly regulated by the production of chemokines in the inflamed microenvironment and switching of expression of specific chemokine receptors on the surface of the DCs (20). Immature DCs express a wide variety of chemokine receptors, including CCR1 (receptor for CCL5 [RANTES]), CCR2 (receptor for CCL2, 8, 7, and 13 [MCP-1, -2, -3, and -4]), CCR3 (receptor for CCL11 [eotaxin]), CCR5 (receptor for CCL3 and 4 [MIP-1{alpha} and ß] and CCL5), and CCR6 (receptor for CCL20 [23, 27]). As a result of the expression of these receptors and interaction with their respective chemokine ligand, immature DCs are rapidly recruited into tissues undergoing inflammatory responses. CCL20 is an unusual CC chemokine recently identified through bioinformatics (28), as it is a unique functional chemokine ligand for CCR6 (24). CCR6 is expressed by a number of immunomodulatory cells, including CD1a+ Langerhans precursor cells (29), as well as mature Langerhans cells. Thus, CCL20 has been shown to be particularly important in CD1a+ DC (Langerhans precursor cell) migration in vitro (24) and has been localized to inflamed epithelium in the skin and tonsil crypts (11), which are also enriched with DC. Recruitment of circulating CD11c+CD1a+ imDCs into the epidermis of the skin and maturation to Langerhans cells is controlled by release of epithelial CCL20 (11, 15, 24). Evidence from nonpulmonary tissues suggest that epithelial Langerhans cells capture antigen and are induced to a mature state by inflammatory cytokines in the surrounding milieu, such as TNF-{alpha}, IL-1, and, in particular, GM-CSF, as well as bacterial or viral products, such as LPS, CpG, or double-stranded RNA (20). Subsequent upregulation of CCR4, CCR7, and CXCR4 (30), alongside downregulation of CCR1, CCR5, and CCR6 (30) and expression of costimulatory molecules such as CD80 and CD86 enable migration to the lymph nodes and activation and polarization of T cells.

This investigation and others suggest a minor role for other chemokines in CD1a+ DC migration, including CCL2, CCL3, CCL5, and CCL7 (11, 22). With respect to CCL5, our previous study of ATII cell chemokine production suggests that it is not a significant ATII cell chemokine (13). However, it is possible that other types of stimulant, for example IFN-{gamma}, may upregulate ATII cell CCL5 expression; furthermore, human alveolar type I cells have been shown to secrete CCL5 (31) and may contribute to DC recruitment. Our observation of CCL-2–induced CD1a+ DC migration is in contrast to that of Dieu-Nosjean and coworkers (11); as the degree of migration was comparatively low (~ 15%), it is not clear how significant this may be in vivo, despite our previous observation that ATII cells can release high levels of CCL2 (13). The significance of ATII cell release of chemokines other than CCL20 in Langerhans cell recruitment to the respiratory unit remain to be established.

In summary, we have shown that LPS-stimulated human ATII cells release comparatively high levels of CCL20. In addition, the migratory response of peripheral blood monocyte–derived CD1a+ DC to ATII cell conditioned media was related to the amount of CCL20, but did not correlate with the levels of other chemokines, CCL2, CCL3, CCL5, CXCL1, and CXCL8. CD1a+ DCs also migrated in response to recombinant human CCL20 in a dose-dependent manner. In contrast, little migration was seen toward conditioned media from macrophages that were isolated from the same tissue. This supported our hypothesis that the ATII cell plays a key role in DC trafficking to the respiratory unit, and that this was mostly due to CCL20 production. It also further implicates a close functional relationship between the Langerhans DC and the pulmonary epithelium, as previously noted for other tissues by other groups (11, 32).


   Footnotes
 
This work was supported by an AstraZenenca Ph.D. studentship (A.J.T.).

Conflict of Interest Statement: A.J.T. received £68,000 GBP (U.S. $119,000) from AstraZeneca PLC as a Ph.D. studentship; P.G. has no declared conflicts of interest; A.Y. is an employee of AstraZeneca Pharmaceuticals; and T.D.T. has no declared conflicts of interest.

Received in original form June 18, 2004

Received in final form November 30, 2004


   References
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

  1. Hart DN. Dendritic cells: unique leukocyte populations which control the primary immune response. Blood 1997;90:3245–3287.[Free Full Text]
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