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Published ahead of print on March 15, 2007, doi:10.1165/rcmb.2006-0235OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 37, pp. 75-84, 2007
© 2007 American Thoracic Society
DOI: 10.1165/rcmb.2006-0235OC

Airway Epithelial IL-15 Transforms Monocytes into Dendritic Cells

Nicolas Regamey*, Carolina Obregon*, Sylvie Ferrari-Lacraz, Coretta van Leer, Marc Chanson, Laurent P. Nicod and Thomas Geiser

Pediatric Respiratory Medicine, and Respiratory Medicine, University Hospital of Berne, Berne; Division of Immunology and Allergy, and Laboratory of Clinical Investigation III, Department of Pediatrics, University Hospitals, Geneva, Switzerland

Correspondence and requests for reprints should be addressed to Prof. L. P. Nicod, Division of Respiratory Medicine, University Hospital of Berne, Freiburgstr. 15, CH-3010 Berne, Switzerland. E-mail: Laurent.Nicod{at}insel.ch


   Abstract
Top
 Abstract
CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
IL-15 has recently been shown to induce the differentiation of functional dendritic cells (DCs) from human peripheral blood monocytes. Since DCs lay in close proximity to epithelial cells in the airway mucosa, we investigated whether airway epithelial cells release IL-15 in response to inflammatory stimuli and thereby induce differentiation and maturation of DCs. Alveolar (A549) and bronchial (BEAS-2B) epithelial cells produced IL-15 spontaneously and in a time- and dose-dependent manner after stimulation with IL-1beta, IFN-{gamma}, or TNF-{alpha}. Airway epithelial cell supernatants induced an increase of IL-15R{alpha} gene expression in ex vivo monocytes, and stimulated DCs enhanced their IL-15R{alpha} gene expression up to 300-fold. Airway epithelial cell–conditioned media induced the differentiation of ex vivo monocytes into partially mature DCs (HLA-DR+, DC-SIGN+, CD14+, CD80–, CD83+, CD86+, CCR3+, CCR6+, CCR7–). Based on their phenotypic (CD123+, BDCA2+, BDCA4+, BDCA1–, CD1a–) and functional properties (limited maturation upon stimulation with LPS and limited capacity to induce T cell proliferation), these DCs resembled plasmacytoid DCs. The effects of airway epithelial cell supernatants were largely blocked by a neutralizing monoclonal antibody to IL-15. Thus, our results demonstrate that airway epithelial cell–conditioned media have the capacity to differentiate monocytes into functional DCs, a process substantially mediated by epithelial-derived IL-15.

Key Words: interleukin-15 • airway epithelial cells • dendritic cells • human studies



   CLINICAL RELEVANCE
Top
 Abstract
 CLINICAL RELEVANCE
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
We show that IL-15 produced by airway epithelial cells induces the differentiation and maturation of monocytes into functional dendritic cells. This indicates an important role of epithelial IL-15 in innate and adaptive lung immunity.

 
Dendritic cells (DCs) are highly specialized antigen-presenting cells (APCs) involved in the initiation and regulation of innate and adaptive immune responses. They are capable of capturing and processing antigens, and of presenting their peptides to naive T lymphocytes after migration into draining lymph nodes (1).

The dense superficial network of airway mucosal DCs is ideally positioned for the interception of any potential antigen in the inhaled air. Pulmonary DCs are distributed along the airways and throughout the pulmonary interstitium in close proximity to overlying epithelial cells. They are present there in a resting state with a high capacity to sense, sample, and process incoming antigens, but only a limited ability to prime naive T cells (2, 3). Upon encounter with microbial, proinflammatory, or T cell–derived stimuli, these immature DCs then undergo characteristic phenotypic and functional changes, a process referred to as maturation. Mature DCs have a reduced phagocytic activity, display large amounts of processed antigens on cell-surface MHC molecules along with T cell costimulatory factors, and secrete immunostimulatory cytokines. Mature DCs also acquire the capacity to migrate to T cell areas of draining secondary lymphoid organs, where they interact with T cells and promote T cell clonal expansion and differentiation.

Even in the absence of overt inflammation, DCs or their precursors are constantly being recruited from the blood into the lung, as first demonstrated by Holt and coworkers (4). However, it is not clear yet whether DCs are recruited from the blood in a differentiated form or as early precursors. DCs could be directly recruited by means of appropriate pulmonary chemokine signals (5). Alternatively, monocytes or CD34+ monocytic DC precursors could first be attracted from the blood into the lung and subsequently differentiate into DCs under the influence of cytokines secreted by resident pulmonary cells (6, 7). The mechanisms of DC differentiation from monocytes or monocytic DC precursors are incompletely understood, and current experimental methods for generating immature DCs commonly use a combination of granulocyte macrophage colony-stimulating factor (GM-CSF) and IL-4 (8, 9).

IL-15 is a multipotent cytokine that shares many biological activities with IL-2, although the sequence of the IL-15 gene shows no homology with the IL-2 gene (10). Both cytokines bind a specific {alpha} subunit, and they share the same beta and {gamma} common receptor subunits for signal transduction (11). IL-15 is produced by various cell types, including epithelial cells in response to environmental stimuli and infectious agents, but not by T lymphocytes, which are the major source of IL-2. IL-15 has been shown to stimulate proliferation, activation, and recruitment of T cells, B cells, and natural killer (NK) cells, and to play a critical role in the functional maturation of DCs and macrophages (12, 13). It has also recently been shown to induce the differentiation of DCs from human peripheral blood monocytes and CD34+ progenitor cells (14–16).

Based on the proximity of epithelial cells and DCs in the lung, we hypothesized that airway epithelial cells are capable of secreting IL-15, and that IL-15 in airway epithelial cell–conditioned media induces the differentiation of ex vivo peripheral blood monocytes into DCs.

A subset of the results of this study has been published in abstract form (17).


   MATERIALS AND METHODS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Cell Cultures
Human A549 (ATCC#CCL185) alveolar epithelial cells were grown in RPMI 1640 medium (Gibco, Paisley, UK) supplemented with 10% heat–inactivated FCS, 2 mM glutamine, 100 U penicillin, and 100 U streptomycin per milliliter, referred to as RPMI complete culture medium (RPMI-CCM). Human BEAS-2B (ATCC#CRL-9609) bronchial epithelial cells were grown in DMEM medium (Gibco) supplemented with 10% heat–inactivated FCS, 2 mM glutamine, 100 U penicillin, and 100 U streptomycin per milliliter (Gibco), referred to as DMEM complete culture medium (DMEM-CCM).

Activation Protocols
A549 and BEAS-2B epithelial cells were seeded in 6-well plates at 105 cells/well and incubated for 48 h to reach confluence in complete culture medium (RPMI-CCM respectively DMEM-CCM). The cells were then washed twice with PBS and incubated for different lengths of time in 500 µl serum-free culture medium containing various concentrations of human recombinant IL-1beta (R&D Systems, Abingdon, UK), IFN-{gamma}, TNF-{alpha} (Roche Diagnostics, Mannheim, Germany), H2O2 (Sigma-Aldrich, Buchs, Switzerland), superoxide (Formula 5 nmol/min/ml) produced by the reaction of xanthine oxidase on xanthine (XaXO) (18), Escherichia coli 0111:B4 LPS (Sigma-Aldrich), purified Salmonella typhimurium Flagellin (Apotech, Lausen, Switzerland), or of a lysate of human granulocytes from healthy blood donors.

IL-15 Enzyme-Linked Immunosorbent Assay
IL-15 in the culture supernatants of airway epithelial cells was determined using a commercial sandwich enzyme-linked immunosorbent assay (ELISA; R&D Systems) with a detection threshold of 1.0 pg/ml. To measure intracellular IL-15 concentration, the cells were lysed in 500 µl PBS with 0.1% Triton-X (Sigma-Aldrich), containing 1 mM PMSF (Sigma-Aldrich) and 0.2 U/ml aprotinin (Bayer AG, Zürich, Switzerland). The lysates were centrifuged at 5,000 x g and the supernatants used for ELISA.

Isolation of Alveolar Macrophages
Alveolar macrophages were isolated from bronchoalveolar lavage (BAL) fluid obtained from patients undergoing diagnostic workup because of suspected bronchial carcinoma. BAL was performed in the middle lobe or lingula, opposite the suspected tumor, by instillation of 3 x 50 ml of 0.9% saline. Between 50 and 60% of the instilled fluid was recovered. All patients gave written informed consent, and the protocol was approved by the local ethics committee. BAL fluid was centrifuged for 5 min at 250 x g, and alveolar macrophages were purified by adherence on plastic in RPMI-CCM for 1 h.

RNA Isolation and Real-Time PCR
Total cellular RNA was isolated from monocytes, DCs, and alveolar macrophages by lysing the cells with TRIzol reagent (Life Technologies, Basel, Switzerland) according to the manufacturer's instructions. The concentration of the RNA samples was determined with a Beckman Coulter Spectrophotometer DU 640. One microgram of RNA was treated with DNase to eliminate any contaminating genomic DNA and subsequently reverse-transcribed as previously described (19). The quality of the reverse transcription was controlled for the expression of the housekeeping gene glyceraldehyde-3' phosphate dehydrogenase by PCR (20). Subsequently, the relative abundance of IL-15 and its specific receptor (IL-15R{alpha}) were determined by TaqMan Real-time PCR analysis using the ABI Prism 7900 Sequence detection instrument. To quantify the levels of cDNA, the expression of IL-15 or IL-15R{alpha} were normalized against the housekeeping gene 18S. Data were expressed as relative fold difference between cDNA of the study samples and a calibrated sample (i.e., control monocytes) (21). IL-15, IL-15R{alpha}, and 18S primer-probe sets were purchased from Applied Biosystems (Foster City, CA).

Monocyte Isolation and Differentiation to DCs
Monocytes were prepared as described (22). In brief, peripheral blood monocytes (PBMC) of healthy human donors were isolated by Ficoll-Hypaque density gradient centrifugation, resuspended in RPMI-CCM supplemented with 2 µg/ml polymyxin B sulfate (Sigma-Aldrich), and incubated for 40 min at 4°C under rotation for aggregation, followed by 10 min of incubation on ice. Pellets of aggregated enriched monocytes were further separated from nonaggregated PBMC by a gradient of FCS and another 10 min of incubation on ice. The monocyte-enriched fraction was incubated overnight with sheep red blood cells (SRBC; bioMérieux, Geneva, Switzerland) to deplete contaminant lymphocytes by rosetting. Monocytes were then isolated by Ficoll-Hypaque density gradient centrifugation. Differentiation of DCs from monocytes was performed by culture in the presence of GM-CSF (10 ng/ml) and IL-4 (10 ng/ml) for 5 d as previously described (8), or by culture in airway epithelial cell supernatants (ESN) obtained by pooling the supernatants of A549 cells stimulated for 24 h in the presence of 10 ng/ml IL-1beta as described above. On Day 3, the culture medium was replaced with fresh medium or ESN. Recombinant IL-15 (50 pg/ml) and rIL-1beta (10 ng/ml), as well as neutralizing monoclonal antibodies to IL-15 (1 µg/ml) and GM-CSF (1 µg/ml) (all from R&D Systems), were added in some experiments. DCs were stimulated with LPS (100 ng/ml) for 24 h in some experiments.

Cell Phenotypic Analysis by Flow Cytometry
Cell surface immunophenotype was analyzed by staining the DCs with phycoerythrin- or fluorescein isothiocyanate (FITC)-labeled monoclonal antibodies against CD1a, CD14, CD68 (Becton and Dickinson, Allschwil, Switzerland), CD11c (Ancell, Bayport, MN), CD80, CD83 (Chemicon, Hampshire, UK), CD86 (Pharmingen, Allschwil, Switzerland), CD123, BDCA1, BDCA2, BDCA3, BDCA4 (Miltenyi, Bergisch Gladbach, Germany), HLA-DR (Dako, Baar, Switzerland), Langerin (Immunotech, Beckman Coulter, Nyon, Switzerland), CCR3, CCR6, CCR7, and DC-SIGN (R&D Systems). The cells were harvested, resuspended in PBS to obtain a single cell suspension, washed, and incubated for 45 min at 4°C in the dark with the staining antibody. For some experiments, 20 µl FcR blocking reagent per 107 cells were added (Miltenyi). Unbound antibody was removed by washing the cells three times in staining buffer (1% BSA and 0.1% NaN3 in PBS) at 4°C. The cells were then resuspended in staining buffer and analyzed with the FACScan (Becton Dickinson).

Dextran-Uptake Assay
The endocytic activity of DCs was determined by incubating 1 x 106 cells with FITC-dextran (Molecular Probes, Leiden, the Netherlands) at the final concentration of 1 mg/ml for 60 min at 37°C (2). As a control, a portion of DCs was incubated with FITC-dextran on ice. The cells were washed with cold RPMI-CCM containing 0.01% NaN3 and analyzed by flow cytometry using FACScan.

Antigen Presentation Assay
Freshly isolated monocytes were cultured in the presence of IL-4/GM-CSF (IL-4/GM-CSF-DCs) or ESN (ESN-DCs) for 2 or 5 d. For the soluble antigen presentation assay, the cells were then pulsed with 0.5 µg/ml tetanus toxoid (TT) for 3 h and cultured in increasing numbers with 1.5 x 105 autologous lymphocytes for 5 d. For the allo-antigen presentation assay, the cells were cultured in increasing numbers with 1.5 x 105 allogenic lymphocytes for 5 d. [Methyl-3H]-thymidine (0.5 µCi/well) was added for the last 18 h.

Statistical Analysis
Results are expressed as mean ± SEM. Statistical significance was determined using Student's t test. Differences were considered significant if P was < 0.05.


   RESULTS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
A549 and BEAS-2B Airway Epithelial Cells Produce IL-15 Spontaneously and upon Stimulation with Proinflammatory Cytokines
First, we investigated whether human bronchial and alveolar epithelial cells produce IL-15 spontaneously and upon stimulation. IL-15 was detected in A549 alveolar epithelial cells and BEAS-2B bronchial epithelial cells, both intracellular and in the cell-free supernatants (Figures 1A and 1B). Upon stimulation with IL-1beta, TNF-{alpha}, and IFN-{gamma}, a marked and significant increase of IL-15 protein was observed in A549 cells. In contrast IFN-{gamma}, but not IL-1beta or TNF-{alpha}, induced the synthesis and secretion of IL-15 in BEAS-2B cells. IL-15 protein production was increased by nearly 5-fold upon stimulation with 10 ng/ml IL-1beta on A549 epithelial cells (76.9 pg/ml versus 17.5 pg/ml, P < 0.01), and by nearly 5-fold upon stimulation with 250 U/ml IFN-{gamma} on BEAS-2B epithelial cells (93.1 pg/ml versus 19.1 pg/ml, P < 0.01) compared with serum free medium control. Synthesis and secretion of IL-15 protein was dose-dependent in A549 cells for IL-1beta and TNF-{alpha} and in BEAS-2B cells for IFN-{gamma} (Figures 1C–1E). A marked increase of IL-15 protein synthesis and secretion was observed already at very low IL-1beta and IFN-{gamma} concentrations (10 pg/ml and 10 U/ml, respectively), whereas an effect of TNF-{alpha} appeared only at a concentration of 1 ng/ml.


Figure 1
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  		Figure 1. Alveolar A549 and bronchial BEAS-2B epithelial cells produce IL-15 spontaneously and upon stimulation with pro-inflammatory cytokines. (A–E) Cells were incubated for 48 h in serum-free culture medium alone or supplemented with IL-1beta, TNF-{alpha}, or IFN-{gamma} at the concentrations indicated. IL-15 was measured by ELISA in cell-free supernatants and cell lyzates. Dose-dependent IL-15 synthesis and release were observed in both A549 and BEAS-2B airway epithelial cells. Open bars, supernatants; solid bars, cell lysates. Data from 4–6 independent experiments are presented as mean ± SEM, **P < 0.01 compared with medium control. (F–H) Time-course analysis of epithelial IL-15 protein production and secretion. Mean values from three independent experiments are shown.

 
Time-course experiments showed a continuous increase of IL-15 protein production both in A549 and in BEAS-2B cells over time (Figures 1F–1H). Irrespective of the type of stimulation and the cell type used, the protein was detectable in higher amounts intracellularly within the first 24 h after stimulation, whereas its level increased markedly in ESN after 24 h, indicating the release of intracellular IL-15 into the cell supernatants.

Stimulation of the airway epithelial cells with oxidative stress (superoxide Formula at the concentration of 5 nmol/min/ml, or H2O2 at the concentrations of 100 µMol and 200 µMol), with bacterial products (flagellin or LPS at a concentration of 100 ng/ml), or with a lysate of human granulocytes did not result in an increased synthesis and production of IL-15 protein, although the cell viability (as shown with trypan blue staining) was 100% after stimulation (data not shown).

IL-15 and IL-15R{alpha} Gene Expression Are Differentially Up-Regulated in Monocytes and DCs Compared with Alveolar Macrophages upon Stimulation with LPS and Airway Epithelial Cell Supernatants
We then determined the expression of IL-15 and IL-15R{alpha} in freshly isolated monocytes, in DCs differentiated with IL-4/GM-CSF, and in alveolar macrophages obtained from BAL fluid. In monocytes, a > 10-fold up-regulation of IL-15R{alpha} cDNA expression was observed upon LPS stimulation, with a maximum at 6 h after stimulation (Figure 2A). In DCs, IL-15 and IL-15R{alpha} cDNA expression increased upon stimulation with LPS as early as 3 h after stimulation up to 10-fold for IL-15 and 300-fold for IL-15R{alpha}. Interestingly, almost no increase of IL-15R{alpha} cDNA expression in alveolar macrophages and no modification of IL-15 expression in monocytes and alveolar macrophages were observed. In contrast, under the same conditions, TNF-{alpha} gene expression was highly increased in LPS-stimulated alveolar macrophages (positive control). Thus alveolar macrophages barely express both IL-15 and IL-15R{alpha}.


Figure 2
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  		Figure 2. DCs, but not alveolar macrophages, up-regulate IL-15 and IL-15R{alpha} upon stimulation with LPS and airway epithelial cell supernatants. (A) Monocytes (Mo), DCs, and alveolar macrophages (AM) were cultured in complete culture medium with or without the addition of LPS (100 ng/ml) for up to 24 h. Total RNA was then extracted for real-time PCR analysis. The expression of the target genes (IL-15 or IL-15R{alpha}) was normalized to the housekeeping gene 18S. Data are presented as relative fold difference between cDNA of the study samples and a calibrated sample (control monocytes, DCs or alveolar macrophages at start of the experiment). The graph shows one of three independent experiments with similar results (values ± SEM of triplicate sample testing). (B) Same experiment with monocytes cultured in complete medium alone, in medium supplemented with LPS (100 ng/ml), or in ESN for up to 24 h.

 
We then studied the effect of ESN on IL-15 and IL-15R{alpha} gene expression in monocytes. As expected, ESN induced an increase in IL-15R{alpha} gene expression in monocytes similar to the increase in IL-15R{alpha} gene expression obtained after stimulation with LPS (Figure 2B). However, as for stimulation with LPS, no modification of IL-15 expression in monocytes was observed upon stimulation with ESN (data not shown).

IL-15 in Airway Epithelial Cell–Conditioned Media Transforms Monocytes into DCs
We subsequently examined whether ESN induce the differentiation of monocytes into DCs, with a particular focus on the role of IL-15. Monocytes cultured in ESN aggregated within 24 h, revealed protruding veils suggestive of DCs within 2 d of culture, and expressed the DC-specific markers CD83 and CD86 (Figure 3A). Time-course experiments showed that the expression of these markers was highest after 2–3 d of culture, with a subsequent decrease toward Day 5 (data not shown). However, ESN did not change the expression level of CD14 or CD80, thereby indicating that ESN-generated DCs were only partially mature. Neutralization of IL-15 with a monoclonal antibody largely blocked ESN-induced CD83 and CD86 up-regulation. Recombinant IL-15 alone, at a concentration similar to that found in ESN (50 pg/ml), also induced CD83 and CD86 up-regulation on DCs, although to a lesser extent than ESN. However, recombinant IL-1beta, at the same concentration as that used to stimulate the airway epithelial cells (10 ng/ml), had no effect on CD83 and CD86 expression on DCs (data not shown). Neutralization of GM-CSF did not inhibit the increased CD86 expression induced by ESN, while it partially blocked CD86 up-regulation in monocytes cultured in IL-4/GM-CSF (Figure 3B). Thus CD83 and CD86 up-regulation in ESN-derived DCs was largely due to IL-15, but not to exogenous IL-1beta or any GM-CSF possibly present in the ESN.


Figure 3
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  		Figure 3. Airway epithelial cell supernatants induce the differentiation of human monocytes into CD83/CD86-positive dendritic cells. Human ex vivo monocytes were cultured under various conditions and the expression of cell surface receptors was measured by FACS analysis. (A) Culture for 5 d in medium alone, in medium supplemented with IL-4 and GM-CSF, in ESN, or in ESN with addition of a neutralizing monoclonal antibody to IL-15 (ESN+{alpha}-IL-15). Data are represented as delta MFI values (mean fluorescence intensity relative to isotype control) ± SEM from 4–6 independent experiments. *P < 0.05 compared with medium alone; {ddagger}P < 0.05 compared with ESN (only indicated if significant). (B) Culture for 5 d in medium supplemented with IL-4 and GM-CSF, or in ESN with or without the addition of a neutralizing monoclonal antibody to GM-CSF ({alpha}GM-CSF). The results of one experiment representative of the three independent experiments performed are shown. (C) Culture for 2 or 5 d in medium supplemented with IL-4 and GM-CSF or in ESN. The graph shows one of three independent experiments with similar results. LC = Langerhans cell.

 
To identify the subset of ESN-generated DCs, the surface markers CD11c and BDCA1 (myeloid DCs), CD123 and BDCA2 (plasmacytoid DCs), and CD1a (Langerhans cell-type) were analyzed (Figure 3C). IL-4/GM-CSF–derived DCs showed a high expression of CD11c and CD1a, as well as a moderate expression of BDCA1 and CD123. In contrast, ESN-derived DCs showed a high expression of CD11c and CD123, as well as a moderate expression of BDCA2, but no expression of BDCA1 and CD1a. Therefore, monocytes cultured in ESN gave rise to DCs with both myeloid (CD11c+) and plasmacytoid (CD123+, BDCA2+) features. We will subsequently call these cells ESN-DCs by contrast to IL-4/GM-CSF-DCs. To further characterize ESN-DCs, additional surface receptors were analyzed (Figure 4). The expression of the DC-specific receptor DC-SIGN was high on both IL-4/GM-CSF-DCs and ESN-DCs, confirming that these cells were DCs. ESN-DCs were both BDCA3- and BDCA4-positive and had some features of interstitial DCs (CD68+), while IL-4/GM-CSF-DCs were only BDCA4-positive. Both IL-4/GM-CSF-DCs and ESN-DCs expressed high levels of CCR3 (not shown), but only ESN-DCs expressed CCR6, a chemokine receptor typically expressed by immature DCs homing to epithelia (23). Thus ESN-DCs had phenotypic characteristics of plasmacytoid DCs (CD123+, BDCA2+, BDCA4+) but shared some characteristics of myeloid (CD11c+, BDCA3+) and interstitial DCs (CD68+).


Figure 4
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  		Figure 4. Airway epithelial cell supernatants-derived dendritic cells have myeloid and plasmacytoid phenotypic characteristics. Ex vivo monocytes were cultured for 3 d in medium supplemented with IL-4 and GM-CSF (red lines) or in airway epithelial cell supernatants (ESN, blue lines) and the expression of cell surface receptors was measured by FACS analysis. The results of one experiment representative of three independent experiments are shown.

 
Airway Epithelial Cell Supernatant-Derived DCs Are Functional
We then tested whether ESN-DCs had the capacity to take up dextran, which is a hallmark of human lung DCs (2). Indeed, a 3-fold increase (from 15% to 50%) in dextran uptake was measured in ESN-DCs compared with nonstimulated monocytes (Figure 5A). This effect was largely abrogated in the presence of a neutralizing antibody to IL-15. However, neutralization of GM-CSF did not inhibit dextran uptake neither in ESN-DCs nor in IL-4/GM-CSF-DCs, indicating that dextran uptake was not GM-CSF dependent (Figure 5B). A time-course analysis of dextran uptake revealed that for both IL-4/GM-CSF– and ESN-DCs, the capacity of dextran uptake reached a maximum already after 1–3 d of culture, with a subsequent decrease as of Days 4–5 (Figure 5C).


Figure 5
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  		Figure 5. Airway epithelial cell supernatants-derived DCs are functional as measured by dextran endocytosis. Ex vivo monocytes were cultured under various conditions and dextran endocytosis was measured by FACS analysis. (A) Culture for 5 d in medium alone, in medium supplemented with IL-4 and GM-CSF, in ESN, or in ESN with addition of a neutralizing monoclonal antibody to IL-15 (ESN+{alpha}-IL-15). Values are presented as mean ± SEM from three independent experiments. *P < 0.05 compared with medium alone (only indicated if significant). (B) Culture for 5 d in medium supplemented with IL-4 and GM-CSF, or in ESN with or without the addition of a neutralizing monoclonal antibody to GM-CSF ({alpha}GM-CSF). The percentage of positive cells is indicated. The graph shows one of three independent experiments with similar results. (C) Time-course analysis of dextran endocytosis for monocytes cultured in medium supplemented with IL-4/GM-CSF and in ESN. Values are means ± SEM of data obtained from three independent experiments.

 
We then evaluated the ability of ESN-DCs to present antigens and to induce T cell proliferation. As shown in Figure 6A, DCs generated after 2 d of culture in ESN were as efficient in inducing autologous T cell proliferation as DCs generated after 2 d in IL-4/GM-CSF–supplemented medium. ESN-DCs generated after 5 d of culture were, however, slightly less efficient in presenting antigens than IL-4/GM-CSF-DCs, a finding in accordance with the observation that co-stimulatory molecule CD86 was maximally up-regulated on ESN-DCs after 2 d of culture, with a subsequent decrease. In contrast, ESN-DCs failed to present alloantigens (Figure 6B).


Figure 6
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  		Figure 6. Airway epithelial cell supernatants-derived DCs induce autologous T cell proliferation but fail to present alloantigens to T cells. IL-4/GM-CSF–derived and ESN-derived DCs were evaluated at Days 2 and 5 for their ability to present antigens. (A) Increasing numbers of DCs (pulsed or not with TT for 3 h) were cultured with autologous T cells, and proliferation was measured by thymidine incorporation. (B) Increasing numbers of DCs were cultured with heterologous T cells, and proliferation was measured by thymidine incorporation. One of two independent experiments with similar results is shown.

 
Airway Epithelial Cell Supernatant-Derived DCs Are Not Fully Mature after LPS Stimulation
We finally tested the ability of DCs to mature after stimulation with LPS. In contrast to IL-4/GM-CSF-DCs, where up-regulation of the accessory molecules CD80, CD83, CD86, and of the maturation marker CCR7 were observed, ESN-DCs only showed a modest up-regulation of CD80 upon stimulation with LPS (Figure 7).


Figure 7
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  		Figure 7. Airway epithelial cell supernatants-derived DCs are not able to fully mature after LPS stimulation. IL-4/GM-CSF–derived and ESN-derived DCs (black lines) were stimulated with LPS (100 ng/ml) for 24 h (blue lines), and the expression of cell surface receptors was measured by FACS analysis. The results of one experiment representative of three independent experiments are shown.

 

   DISCUSSION
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
In the present study, we demonstrate that airway epithelial cell–conditioned media have the capacity to differentiate ex vivo monocytes into DCs, a process substantially mediated by epithelial-derived IL-15.

Mucosal surfaces of the lung are intensely exposed to a wide variety of inhaled antigens. Recent research has revealed that mucosal immune homeostasis is tightly regulated by the crosstalk between epithelial cells and DCs (24, 25). Interactions between epithelial cells and DCs have also been shown to play a critical role in determining the qualitative characteristics of adaptive immune system maturation (26). Thus the airway epithelium has been recognized not only as a physical barrier but also as a modulator of airway inflammation (26). Airway epithelial cells are capable of synthesizing a variety of proinflammatory cytokines or chemokines, such as GM-CSF, IL-1beta, IL-6, IL-8, TGF-beta, RANTES, or eotaxin.

IL-15 mRNA up-regulation upon inflammatory stimuli has been shown earlier in airway epithelial cell lines. However, the expression of IL-15 is regulated at both the transcriptional and the translational levels (27–29). The study of IL-15 protein production and secretion by airway epithelial cells has been difficult due to the low level of IL-15 protein in supernatants and cell lysates compared with other inflammatory cytokines (27, 30–32). We found that both bronchial and alveolar epithelial cells secrete IL-15 protein in a constitutive manner. The two airway epithelial cell types used in this study however showed that IL-15 protein secretion differs depending on the stimulatory cytokine used. Whereas IL-15 protein secretion was up-regulated by IL-1beta, TNF-{alpha}, and IFN-{gamma} in alveolar epithelial cells, only IFN-{gamma} was able to increase IL-15 protein secretion in bronchial epithelial cells, although it has been shown that these cells are responsive to both IL-1beta, and TNF-{alpha} under other conditions (33). This finding is in accordance with those of Ge and colleagues, who showed that IL-15 protein up-regulation was achieved upon stimulation with IFN-{gamma}, but not upon stimulation with other inflammatory cytokines in bronchial epithelial cells (34).

Muro and coworkers evaluated the expression of IL-15 mRNA and protein in human bronchial biopsy specimens (35). They found an increased number of IL-15–positive cells in biopsies from patients with sarcoidosis, tuberculosis, and chronic bronchitis compared with biopsies from patients with bronchial asthma and control subjects, suggesting an association of IL-15 expression with Th1-mediated chronic inflammatory diseases of the lung. Interestingly, the majority of IL-15–immunoreactive cells in their study were subepithelial macrophages and elastase-positive neutrophils, but unfortunately the authors did not examine the epithelium in their bronchial biopsies. However, Liu and coworkers recently reported that IL-15 is highly expressed in the intestinal mucosa of patients with inflammatory bowel diseases, supporting the epithelial origin of IL-15 in conditions other than lung diseases (36).

Previous studies have shown that recombinant IL-15 in high concentrations (100–300 ng/ml), alone or in combination with GM-CSF, induces a differentiation of human monocytes or CD34+ hemopoietic precursor cells into partially mature DCs (14–16, 37). Compared with "classical" DCs generated from monocytes with IL-4 and GM-CSF, or from CD34+ progenitor cells with a combination of GM-CSF, IL-4, FTL3L, and TNF-{alpha}, these DCs were equally able to undergo maturation after interaction with LPS and CD40L, and functionally similar in their ability to prime T cells. While some authors have described that IL-15– or IL-15/GM-CSF–derived DCs had features of Langerhans cells with high expression of CD1a (14, 15), this has not been found by others (16). Here we show that, under physiological conditions, epithelial IL-15 induces a differentiation of monocytes into functional DCs able to take up antigens, as measured by dextrose endocytosis, and to present antigens, as measured by autologous T cell proliferation. However, the fact that recombinant IL-15 in the picogram range alone had a less pronounced effect than epithelial supernatants, and that blocking antibodies against IL-15 did not completely neutralize the effect of epithelial cell–conditioned media, suggests the involvement of other active mediators contained in these airway epithelial cell supernatants. Interestingly, we found no evidence of a major role of GM-CSF, in spite of its known critical effects on DC recruitment, differentiation, survival, and regulation of immunostimulatory activity. This is in line with previous findings of Saikh and colleagues, who showed that conversion of monocytes to DC by IL-15 does not require GM-CSF (16).

It has recently been shown that IL-15/IL-15R{alpha} can be presented in trans to neighboring cells that express intermediate-affinity IL-15R, which suggests that cell-to-cell contact is needed for full action of IL-15 (38, 39). Such a close contact between epithelial cells and DCs exists in the respiratory mucosa. Therefore, we can hypothesize that airway epithelial cells not only induce a differentiation of monocytes into DCs through soluble IL-15 as shown in this study, but may also induce a maturation of resident DCs through the presentation in trans of the IL-15/IL-15R complex to these cells.

The induction of IL-15 mRNA upon stimulation with LPS in DCs is supported by the findings of Santini and coworkers, who demonstrated that the IL-15 gene is silent in monocytes cultured with GM-CSF and IL-4, whereas type I interferon or LPS induce a significant production of IL-15 when added to the same cultures (40). This suggests that a release of IL-15 by DCs may serve as a positive feedback loop to amplify DC activation and recruitment (41, 42). On the other hand, it has recently been shown that IL-15R{alpha} and IL-2Rbeta chains, but not IL-2R{alpha} or {gamma} chains, are constitutively expressed in bronchial epithelial cells, which argues against an autocrine stimulation of airway epithelial cells, as both IL-2Rbeta and {gamma} chains are required for binding and signaling of IL-15 through the IL-2Rbeta{gamma}/IL-15R{alpha} complex (34).

Demedts and colleagues have recently demonstrated the presence of three previously unidentified DC subsets in human lung digests: myeloid DCs type 1 (mDC1, identified by the expression of BDCA1), myeloid DCs type 2 (mDC2, identified by the expression of BDCA3), and plasmacytoid DCs (pDCs, identified by the expression of BDCA2 and CD123), with divergent roles, both in innate and adaptive immune responses (43, 44).

Through their phenotypic and functional properties, ESN-DCs resemble pDCs. They express the plasmacytoid receptors CD123, BDCA2, and BDCA4 and lack the myeloid receptor BDCA1, as well as CD1a, which has been shown to identify a subset of mDCs in bronchoalveolar lavage fluid (BALF) (45). They also express BDCA3, which is usually considered as a myeloid marker but has recently been shown to be expressed on pDCs in BALF (45). ESN-DCs show a partial mature phenotype with low expression of maturation markers (HLA-DR+, DC-SIGN+, CD14+, CD80–, CD83+, CD86+, CCR7–), which is consistent with what has been described by others for pDCs (43). Interestingly, and in contrast to IL-4/GM-CSF-DCs, they express the chemokine receptor CCR6, which is typically found on immature DCs homing to epithelia (23). Airway epithelial cells have been shown to produce the chemokine CCL20 (MIP-3{alpha}), a major chemoattractant for CCR6-bearing immature DCs (46), and in a model of allergic airway inflammation, CCR6 appeared to mediate the recruitment of DCs into the inflamed lung (47). From a functional point of view, ESN-DCs have the ability to take up antigens but only have a limited capacity of maturation upon stimulation with LPS and of inducing allogenic T cell proliferation. This is in line with the observations of Demedts and coworkers, who showed that in contrast to mDCs, which are strong inducers of T cell proliferation, pDCs hardly induce any T cell proliferation, suggesting that pDCs might be involved in the induction of tolerance (43, 44).

However, although ESN-DCs resemble pDCs described in the human lung, they also share phenotypic features of myeloid (CD11c+, BDCA3+) and interstitial DCs (CD68+). Thus, they may represent an intermediate DC differentiation and maturation stage. It has indeed been proposed that all DC subsets can arise from a common DC precursor in blood, and functional plasticity of human respiratory tract DCs has been demonstrated (48, 49). It can therefore be hypothesized that lung DCs in this intermediate maturation step may become tolerogenic or immunogenic, depending on the local environment. Sporri and colleagues have recently reported that inflammatory mediators alone are insufficient for complete DC differentiation and maturation, and that exposure to pathogen components (pathogen-associated molecular patterns, PAMPs) is critical for priming an appropriate T cell response (50). Our observations that ESN-DCs failed to present alloantigens to T cells, but were efficient in inducing autologous T cell proliferation upon stimulation with tetanus toxoid, corroborate these findings. The plasticity of DCs is also demonstrated in our study by the fascinating observation of the differential regulation of the markers CD123 and BDCA2 after 2 and 5 d, respectively, of culture in ESN (Figure 3c).

In conclusion, we demonstrate (1) that IL-15 is secreted by alveolar and bronchial epithelial cells in a constitutive manner and upon stimulation with proinflammatory cytokines; (2) that monocytes and DCs are IL-15 receptive cells by upregulating IL-15R, in contrast to alveolar macrophages; and (3) that IL-15 in airway epithelial cell–conditioned media induces the differentiation of ex vivo peripheral blood monocytes into partially mature functional DCs with plasmacytoic features. Consequently, the epithelial release of IL-15 by inflammatory stimuli may play an important role in the initiation and maintenance of the immune response in the human lung.


   Acknowledgments
 
The authors are indebted to Monika Stutz, Ursula Gerber, Trinh Cung, Natasha Tetkovic, Karin de Peyer, and Rachel Chicheportiche for their excellent technical assistance.


   Footnotes
 
* These authors contributed equally to this work. Back

This work was supported by grants Nr. 3200-100497 (to T.G.), Nr. 3100-107659 (to L.P.N.), Nr. 3200-066357 (to S.F.-L.), and Nr. 3100-107846 (to M.C.) from the Swiss National Science Foundation.

Originally Published in Press as DOI: 10.1165/rcmb.2006-0235OC on March 15, 2007

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 June 30, 2006

Accepted in final form February 15, 2007


   References
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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