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Published ahead of print on February 1, 2007, doi:10.1165/rcmb.2006-0234OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 36, pp. 669-677, 2007
© 2007 American Thoracic Society
DOI: 10.1165/rcmb.2006-0234OC

Dendritic Cells and Macrophages Form a Transepithelial Network against Foreign Particulate Antigens

Fabian Blank*, Barbara Rothen-Rutishauser* and Peter Gehr

Institute of Anatomy, Division of Histology, University of Bern, Bern, Switzerland

Correspondence and requests for reprints should be addressed to Fabian Blank, Institute of Anatomy, Division of Histology, University of Bern, Baltzerstrasse 2, CH-3000 Bern 9, Switzerland. E-mail: fabian.blank{at}ana.unibe.ch


   Abstract
Top
 Abstract
CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Fine particles (0.1–2.5 µm in diameter) may cause increased pulmonary morbidity and mortality. We demonstrate with a cell culture model of the human epithelial airway wall that dendritic cells extend processes between epithelial cells through the tight junctions to collect particles in the "luminal space" and to transport them through cytoplasmic processes between epithelial cells across the epithelium or to transmigrate through the epithelium to take up particles on the epithelial surface. Furthermore, dendritic cells interacted with particle-loaded macrophages on top of the epithelium and with other dendritic cells within or beneath the epithelium to take over particles. By comparing the cellular interplay of dendritic cells and macrophages across epithelial monolayers of different transepithelial electrical resistance, we found that more dendritic cells were involved in particle uptake in A549 cultures showing a low transepithelial electrical resistance compared with dendritic cells in16HBE14o cultures showing a high transepithelial electrical resistance 10 min (23.9% versus 9.5%) and 4 h (42.1% versus 14.6%) after particle exposition. In contrast, the macrophages in A549 co-cultures showed a significantly lower involvement in particle uptake compared with 16HBE14o co-cultures 10 min (12.8% versus 42.8%) and 4 h (57.4% versus 82.7%) after particle exposition. Hence we postulate that the epithelial integrity influences the particle uptake by dendritic cells, and that these two cell types collaborate as sentinels against foreign particulate antigen by building a transepithelial interacting cellular network.

Key Words: dendritic cells • airway macrophages • cellular interplay • particle translocation • confocal laser scanning microscopy



   CLINICAL RELEVANCE
Top
 Abstract
 CLINICAL RELEVANCE
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
The mechanisms of particle uptake by immune cells of the airway wall shown in our study, and in particular the profound involvement of the highly competent antigen-presenting dendritic cells, constitute important new knowledge of the defense mechanism of the lung.

 
A series of structural and functional barriers (Figure 1a) protect the respiratory system against harmful and innocuous particulate material (1). It includes the surfactant film (2–4), the aqueous surface lining layer including the mucociliary escalator (5), a population of macrophages (professional phagocytes) in the airways and in the alveoli (6, 7), the epithelial layer endowed with tight junctions (TJs) between the cells (8, 9), a population of dendritic cells (professional antigen-presenting cells) inside and underneath the airway epithelium (10, 11), and the basal lamina (basement membrane) (12–14). However, despite the existence of these barriers, respiratory diseases are frequent and increasing (15–17), and more attention has been directed toward elucidating how and when the antigens evade these mechanisms of protection. Inhaled particles first encounter the surfactant film when they are deposited in the airways (3, 4). Surfactant is located at the air–liquid interface of the thin liquid lining layer, which covers the lung epithelium. It reduces the surface tension of the liquid and thus facilitates the displacement of particles into the subphase below the surfactant film. As a result of this process particles may be coated with surfactant or components of it and quickly come into contact with the epithelium and the macrophages located on its apical surface (3, 18, 19). Of particular importance and interest is how inhaled particulate antigens come into contact with dendritic cells, which realize, as sentinels and most competent antigen-presenting cells, a surveillance network in the pulmonary tissues (10, 11). Among the most crucial specialized functions of dendritic cells are the capturing and delivering of antigen to local lymphoid tissues (20), and their unique responsibility is to modulate whether to present sampled antigen in an immunogenic or tolerogenic way (21). Inhaled particulate antigen, which is able to cross the epithelial barrier, is very likely to come into contact with the network of dendritic cells in the airway mucosa (22). Furthermore, it has been shown that large macromolecules, which are not able to overcome the epithelial barrier and are deposited on the luminal surface of the epithelium, are also captured by dendritic cells residing underneath the epithelial wall without affecting the epithelial integrity (20). However, the mechanism by which dendritic cells cross the TJs, which "seal" the airway epithelium at the apical side, is not yet clear (22). Several studies focusing on other epithelial tissues containing dendritic cells suggest two different antigen-capturing mechanisms. First, in gut mucosa subepithelial dendritic cells were reported to be capable of capturing antigens outside the epithelium by extending fine cytoplasmic processes between the epithelial cells and through their TJs (23, 24). This mechanism has also been reported for human nasal mucosa of allergic rhinitis in situ, where processes of dendritic cells were shown to penetrate beyond well-developed epithelial TJs (25). Similar findings were reported in a rat model in which 1–5% of intraepithelial dendritic cells were observed to extend projections into the airway lumen, both under steady-state conditions and during inflammation (26). Second, dendritic cells may transmigrate as whole cells through the airway epithelium to sample antigen in the airway lumen, since several studies with mouse models of asthma have shown increased dendritic cell numbers in bronchoalveolar lavage fluid after allergen exposure (27–29). However, their transepithelial migration has not been visualized so far.


Figure 1
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  		Figure 1. Epithelial airway wall. (a) Barrier components of the airway wall, schematic drawing. The cells involved are: epithelial cells (red), airway macrophages (blue), and dendritic cells (yellow). (b) Triple cell co-culture simulating the airway wall. Numbers in a: 1, the surfactant film; 2, the aqueous surface lining layer including the mucociliary escalator; 3, a population of macrophages (professional phagocytes) in the airways; 4, an epithelium with tight junctions; 5, a population of dendritic cells (professional antigen-presenting cells) in the airways; 6, the basal lamina. A monolayer of epithelial cells was grown on a microporous insert membrane (M) in a two-chamber system. MDM were placed on top of the epithelium while MDDC were placed underneath the insert membrane. Fluorescent 1-µm polystyrene particles were sprayed on the air-exposed co-cultures.

 
Human dendritic cells are rare cells in situ. They represent at most a few percent of the total cell population in a given organ (21, 22), which makes it very difficult to study their mechanism of capturing particulate antigen and their interplay with other cells of the lung barrier like macrophages and epithelial cells in vivo. Furthermore, isolating dendritic cells from tissue samples easily induces activation artefacts, because dendritic cells are exquisitely sensitive to stress signals arising from the environment (21). Therefore we have established a triple cell co-culture model composed of a monolayer of A549 cells, a cuboidal human lung epithelial cell line (30), grown on a microporous membrane, human blood monocyte–derived macrophages (MDM), added on top of the epithelium, and human blood monocyte–derived dendritic cells (MDDC), added underneath the membrane (Figure 1b) (31, 32). The triple cell co-cultures were exposed to air for 1 d, after which fluorescently labeled 1 µm polystyrene particles were nebulized over the epithelial surface (Figure 1b) (32). For examination with laser scanning microscopy (LSM) combined with digital image restoration (which allows the investigations of the relative spatial localisation of several cell layers), the co-cultures were fixed and labeled for the surface markers CD14 (MDM) and CD83 (MDDC). The actin-cytoskeleton of the cells was stained with phalloidin rhodamine. The involvement of MDM and MDDC in particle uptake was quantified using random sampling. Furthermore, co-cultures with A549 epithelia were compared to co-cultures with 16HBE14o epithelia (a human bronchial epithelial cell line [33]). Since 16HBE14o monolayers were reported to have a markedly higher transepithelial electrical resistance (TEER) than A549 monolayers (34), the interplay of MDDC and MDM during particle uptake and particle translocation depending on the tightness of the epithelium was studied.


   MATERIALS AND METHODS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Triple Cell Co-Cultures
Cultures were prepared as previously described (31, 32). Briefly, A549 cells (passages 10–40) were grown on cell culture inserts (surface area of 4.2 cm2, pores with 3.0 µm diameter, high pore density PET membranes for 6-er well plates; BD Biosciences, Basel, Switzerland). For alternative co-cultures the bronchial epithelial cell line 16HBE14o (passage number 2.45–2.80) was used. 16HBe14o cells were maintained in MEM 1x, with Earle's Salts, 25 mM HEPES, without L-Glutamine (Gibco BRL Life Technologies Invitrogen AG, Basel, Switzerland) supplemented with 1% L-Glutamine (LabForce AG, Nunningen, Switzerland), 1% penicillin/streptomycin (Gibco BRL), and 10% fetal calf serum (PAA Laboratories, Lucerna-Chem AG, Lucerne, Switzerland) on transparent BD Falcon cell culture inserts (surface area of 4.2 cm2, pores with 3.0 µm diameter, PET membranes for 6-er well plates; BD Biosciences) treated with fibronectin coating solution containing bovine serum albumin, 0.1 mg/ml (Sigma, Fluka Chemie GmbH, Buchs, Switzerland) + 1% bovine collagen, Type I (BD Biosciences, Basel, Switzerland) + 1% human fibronectin (BD Biosciences) in LHC Basal Medium (Lucerna Chemie AG) as follows. The culture conditions for 16HBE14o cells were the same as for A549 cells. MDM and MDDC were derived from human blood monocytes as already described (31). Briefly, peripheral blood monocytes were isolated from buffy coats (blood donation service) and cultured in the same medium as used for the epithelial cells except for the supplemention of 5% human serum (blood donation service Bern, Switzerland) instead of 10% fetal calf serum. For the generation of MDDC the monocytes were cultured for 7–10 d in medium supplemented with 34 ng/ml IL-4 (Sigma, Fluka Chemie GmbH) and with 50 ng/ml GM-CSF (R&D Systems, Oxon, UK), whereas the MDM were obtained without any additional supplements for 7–10 d. Epithelial cells were cultured for 7 d before MDM were added on top of the epithelial monolayer and MDDC underneath the insert membrane. The triple cell co-cultures were kept overnight in medium supplemented with 1% L-Glutamine, 1% penicillin/streptomycin, and 5% heat-inactivated (pooled) human serum at 37°C in 5% CO2 humidified atmosphere. The next day the medium was removed completely from the upper chamber while 2 ml of medium were kept in the lower well to feed the cultures from the basal side of the insert. The cells were exposed to air for 24 h at 37°C in 5% CO2 humidified atmosphere.

TEER Measurements
TEER was measured in air-exposed 16HBE14o and A549 co-cultures as described earlier (31, 32) with the Millicell-ERS system (MERS 000 01; Millipore AG, Volketswil, Switzerland). Briefly, TEER was measured in the epithelial monocultures, in the co-cultures before and 2, 6, and 24 h after particle exposure. The mean of three measurements per insert was determined and the two measurements were compared. The electrical resistance of filters without cells was subtracted from all samples, and the resistance values were multiplied with the surface area of the inserts (4.2 cm2).

Particle Exposure
After the cultures were exposed to air for 24 h, particles were sprayed on the apical surface of the cultures with a MicroSprayer model IA-1C with a 10" long 0.64-mm tube (PennCentury, Philadelphia, PA). Particle suspension was prepared as follows: fluoresbrite microspheres, plain yellow green with a diameter of 1 µm, were obtained from Polyscience (Brunschwig, Basel, Switzerland). Particles were diluted to a concentration of 6.5 x 105 particles/µl in RPMI 1640 and sonicated for 2 min. For adequate particle distribution on the sample (32), 25 µl (1.5 x 107particles) of particle suspension were sprayed on each air-exposed co-culture (>3.6 x 106 particles/cm2). Cultures were either fixed within 10 min after spraying or incubated for another 4 or 24 h.

Cell Labeling and Fixation
Co-cultures were fixed and stained as previously described (31). Antibodies were diluted in PBS as follows: mouse anti human CD14 1:20 (Clone UCHM-1, C 7673; Sigma), mouse anti-human CD86 1:20 (immature MDDC, Clone HB15e, 36931A; PharMingen, BD Biosciences), mouse anti-human CD83 R-PE-conjugated 1:5 (mature MDDC, Clone HB15e; BD Biosciences), rabbit anti-human occludin 1:20 (71–1500; Zymed, P. H. Stehelin & Cie AG, Switzerland), goat anti-rabbit cyanine-5 1:50 (AP187S; Chemicon, VWR International AG, Life Sciences, Lucerne, Switzerland), goat anti-mouse cyanine 5 1:50 (AP124S; Chemicon), Sytox green Nucleic acid stain (S-7020; Molecular Probes, Juro Supply GmbH, Lucerne, Switzerland), Alexa Fluor 488 phalloidin 1:50 (A12379; Molecular Probes), and rhodamine phalloidin 1:100 (R-415; Molecular Probes). The specificity of the antibodies and the labeling procedure was tested with the following control experiments: use of secondary antibodies only, by using the macrophage-specific antibody CD14 in epithelial-MDDC co-cultures; or by using the dendritic cell–specific antibodies CD83 and CD86 in epithelial-MDM co-cultures. The antibodies were highly specific.

Laser Scanning Microscopy and Image Restoration
A Zeiss LSM 510 Meta with an inverted Zeiss microscope (Axiovert 200M; lasers: HeNe 633 nm, HeNe 543 nm, and Ar 488 nm; Carl Zeiss AG, Feldbach, Switzerland) was used. Image processing and visualization was performed using IMARIS (Bitplane AG, Zurich, Switzerland), a three-dimensional multi-channel image processing software for confocal microscopic images (31, 32). To visualize the labeled MDDC and processes of MDDC or MDM inside the epithelium, a rendering mode was used, which shows the maximum intensity projection (i.e., the maximum intensity of all layers along the viewing direction) of the recorded three-dimensional stack. To illustrate the "luminal" surface of the co-cultures, a shadow projection was applied from different observation angles. For the visualization of three-dimensional data sets, particularly for the localization of particles inside the cells, the surpass module from IMARIS was used, which provides extended functions: the volume rendering, which displays the volume of the entire data set, or the IsoSurface visualization, which is a computer-generated representation of a specific grey value range in the data set. It creates an artificial solid object to visualize the range of interest of a volume object.

Quantification of Cell Numbers and of the Phagocytic Index
For the quantification of cell numbers three independently grown co-cultures (different passages of epithelial cells, with MDM and MDDC from different monocyte isolations) were processed for CLSM as already described above. Confocal Z-stacks of at least three fields of 230 µm x 230 µm x 35–45 µm were taken in each independent sample in a systematic random manner using the laser scanning microscope and a x40 objective lens (oil immersion, NA = 1.3). CD14 (MDM)- and CD86 (MDDC)-positive cells were counted per Z-stack using the forbidden line rule (35). The migration of MDDC across the epithelial monolayer was estimated by counting MDDC profiles on top of the epithelium in each Z-stack and divided by the total number of MDDC profiles counted in the same stack. The migration index of MDDC (MID) was estimated as follows:

Formula
where D(A) represents the dendritic cells on the apical side of the epithelium and D(B) the cells that remained at the basal side.

To estimate the phagocytic index of MDM and MDDC, three independently grown and particle-exposed co-cultures were processed for each time point after exposition (10 min, 4 h, 24 h), random samples were taken, and cell profiles were counted as described above. The phagocytic index (PI) was estimated from:

Formula
where C(p) represents the cell profiles containing particles and C(0) the cell profiles that did not contain particles (36).

Statistics
The statistical analysis of the TEER measurements, the counted cell numbers, and the PI was performed using SigmaStat for Windows (Version 3.10; Systat Software, Inc., Richmond, CA) statistical software. Two groups were compared using Student's t test when sample ranges passed normality test. When the normality test failed, Mann-Whitney Rank Sum Test was applied. P < 0.05 was considered to be significant.


   RESULTS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Morphometric Analysis of the Triple Cell Co-Cultures
A549 and 16HBE14o co-cultures were characterized by estimating cell densities using random sampling (Table 1). Both MDM and MDDC densities did not differ among the two airway wall models (P >= 0.05). The average epithelial surface area occupied by one MDM was between 4,350 and 5,150 µm2. An average area between 2,430 and 2,730 µm2 was occupied by one MDDC.


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  		TABLE 1. NUMBER OF EPITHELIAL CELLS, MDM, AND MDDC IN THE TRIPLE CELL CO- CULTURES

 
16HBE14o epithelial cells built a monolayer of a thickness of around 10 µm and the cells showed an average surface area around 180 µm2. A549 monolayer thickness was about the same as estimated for 16HBE14o cultures, and cells showed an average surface area of 103 µm2. Thus, the cell density of A549 cells was significantly higher than densities of 16HBE14o cells (P <= 0.05).

Estimation of Phagocytic Index
The phagocytic capacity of MDM and MDDC was first compared within the two different culture models (Figure 2). In A549 co-cultures the phagocytic index (PI) increased steadily over time from 12.8% (10 min) to 72.0% (24 h) for MDM, and from 23.9% (10 min) to 46.8% (24 h) for MDDC. In 16HBE14o co-cultures PI also increased steadily over time from 42.8% (10 min) to 87.6% (24 h) for MDM, and from 9.5% (10 min) to 31.5% (24 h) for MDDC. In A549 co-cultures no significant difference in PI between MDM and MDDC could be found 10 min (P >= 0.05) and 4 h (P >= 0.05) after particle exposure. However, after 24 h significantly more MDM than MDDC were involved in particle uptake (P <= 0.01). In 16HBE14o co-cultures MDM had always a higher PI than did MDDC.


Figure 2
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  		Figure 2. Comparison of the phagocytic index of MDM and MDDC in A549- and 16HBE14o Co-cultures. Cultures were fixed 10 min, 4 h, and 24 h after exposition to 1-µm polystyrene particles, and PI of MDM and MDDC was evaluated using random sampling. *Significant difference (P <= 0.05) in PI of MDM in A549 co-cultures versus 16HBE14o co-cultures 4 h and 24 h after particle exposure. §Significant difference (P <= 0.05) in PI of MDDC in A549 co-cultures versus 16HBE14o co-cultures 4 h and 24 h after particle exposure. #Significant difference (P <= 0.05) in PI of MDDC versus MDM 24 h after particle exposure in A549 co-cultures. (Note: In 16HBE14o co-cultures, PI of MDM was always significantly higher compared with the PI of MDDC in the same co-cultures.)

 
In a second step, the phagocytic index of MDM and MDDC of the two co-culture models was compared (Figure 2). In 16HBE14o co-cultures significantly more MDM were involved in particle uptake 10 min (P <= 0.01) and 4 h (P <= 0.05) after particle exposure than MDM in A549 co-cultures. In contrast, significantly more MDDC were involved in particle uptake 10 min (P <= 0.05) and 4 h (P <= 0.01) after particle exposure in A549 co-cultures compared to MDDC in 16HBE14o co-cultures. This indicates an influence of the PI of MDDC on the particle uptake activity of MDM. However, 24 h after particle exposure no significant difference (P >= 0.05) in PI between MDM and between MDDC could be found among the different the co-cultures (Figure 2).

Evaluation of the MID across the Epithelial Monolayer in A549- and 16HBE14o Co-Cultures
The MID across the epithelium was monitored after particle exposition and compared among the two different culture models (Table 2). An MDDC migration to the apical side of the epithelium between 13.3% (24 h) and 17.9% (10 min) was estimated with no significant change over time in A549 co-cultures (P >= 0.05). MID in 16HBE14o co-cultures was between 6.6% (24h) and 11.5% (4h) with no significant change over time (P >= 0.05). However, 10 min (P <= 0.01) and 24 h (P <= 0.05) after particle exposure, more MDDC were found at the apical side of A549 co-cultures than in 16HBE14o co-cultures, indicating a higher migration rate of MDDC to the apical side of the epithelium in A549 co-cultures.


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  		TABLE 2. COMPARISON OF THE MDDC MIGRATION ACROSS THE DIFFERENT EPITHELIAL MONOLAYERS

 
Visualization of Particle Clearance by MDM
Immediately after deposition (samples were fixed within 10 min after particle exposure) of fluorescently labeled polystyrene particles with a diameter of 1 µm, MDM, residing on top of the epithelial surface with a round shape, started to take up the particles (see also Figure 2), as some of them could already be found attached to spherical shaped MDM (Figure 3a). After 24 h there was a visible clearance of particles from the epithelial surface in both culture models; many MDM, which seemed to spread out over the epithelial surface upon particle deposition, were filled with particles (Figure 3b). Some of the MDM were found embedded between epithelial cells, but a part of their surface was still exposed to the "luminal side" (Figure 3c). The same cells extended processes between the epithelial cells toward the basal side and even through the membrane pores to the bottom side of the insert membrane (Figures 3c'–3c'''), where the MDDC are located.


Figure 3
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  		Figure 3. Interplay of MDM with epithelial cells after particle exposure. Clearance of 1-µm particles (white) on top of the epithelium (Ep) (actin, red) by MDM (blue) 0 h (a) and 24 h (b) after particle exposure. (c) MDM embedded in the epithelium (actin, red) 4 h after particle exposure. (c') MDM extended processes through the epithelium and the membrane pores (M, asterisks). (c'' and c''') Extensions of the MDM proceeded tightly between epithelial cells (circles). (a–c''') One-micrometer particles were found on top of the epithelium (arrowheads), either attached to (arrowheads) or inside the MDM (arrows). c–c''' represent the same data set. (a–c) Volume rendering (shadow projection). (c') Surface rendering. (c'') Maximum intensity projection of the xy-viewing direction. (c''') Maximum intensity projection of the xz- viewing direction. Bar is 10 µm in a and b, 5 µm in c'' and c'''.

 
MDDC Capture Particles at the "Luminal Side"
In the absence of particles, MDDC residing underneath the insert membrane were found to reach to the apical side of the epithelium, with small cytoplasmic processes that they pushed up between the A549 cells (Figure 4a). In particle exposed co-cultures MDDC, located underneath the insert membrane, were found to extend long processes through the pores of the insert to spread out directly underneath the epithelium (Figures 4b and 4b'). In addition, they pushed processes up between the epithelial cells into the "luminal space." One-micrometer particles were found inside these processes on top and underneath the epithelium (Figures 4b and 4b'), demonstrating particle translocation through MDDC processes across the epithelium to the basal side. Moreover, in both co-culture models MDDC were found migrating through the insert membrane and through the epithelium (see also Table 2)—that is, between the epithelial cells—finally residing on top of the epithelium, where they collected particles (Figure 4c; Table 2). Nuclear staining demonstrated that these cells moved completely (including the nuclei) through the membrane pores and through the epithelium to the "luminal side" (Figure. 4d). Most of these cells, however, maintained processes between the epithelial cells to the basal side (Figure 4c).


Figure 4
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  		Figure 4. Particle uptake and their translocation across the epithelium by MDDC. (a–d) Co-cultures with A549 cells. (a) In absence of particles, MDDC (yellow) extended processes between epithelial cells (Ep) (actin, red) into the "luminal space" (black asterisks). (b) Twenty-four hours after particle exposure, in presence of particles, an MDDC pushed processes through the membrane pores (M) (white asterisk) underneath the epithelium (actin, red) and between the epithelial cells towards the "luminal space" (black asterisk). (b') Inset I illustrates the apical extension with intracellular 1-µm particles (red, arrow). In inset II, particles (red) are visible inside the MDDC underneath the epithelium (arrows). (c) Twenty-four hours after particle exposure, MDDC (yellow) laying on top of the epithelium (actin, red) with intracellular particles (red, arrows). A process of the MDDC toward the basal side of the epithelium is clearly visible. (d) Twenty-four hours after particle exposure, some MDDC (yellow) migrated as complete cells to the apical side of the epithelium (Ep, red); nuclei (N) are shown in green. Inset I shows a perinuclear part of an MDDC migrating through a membrane pore. Co-cultures with 16HBE14o cells are shown 4 h (e) or 10 min (f) after particle exposure; an MDDC (yellow) is also shown residing underneath the insert membrane (e) and on top of it (f). Both MDDC pushed processes up between epithelial cells (Ep, red) to sample particles (arrows). a (top), b, c (inset), e (inset), and f: maximum intensity projections of xy- and xz viewing directions (particles: white). a (bottom), b', c, d, and e: volume (shadow projection), surface rendering (particles: red). Bars are 10 µm.

 
MDDC Penetrate the TJs of the Epithelium
Independently of the higher TEER of the 16HBE14o monolayer (see below), MDDC in co-cultures with 16HBE14o cells showed a similar interaction with the epithelium by extending processes between the epithelial cells into the "luminal space" to take up deposited particles (Figures 4e and 4f). However, most MDDC cell bodies were seen either underneath the insert membrane (Figure 4e) or between the membrane and the epithelium (Figure 4f). In cultures that were fixed 10 min after particle exposition, some MDDC already contained particles in processes on top of the epithelium (Figure 4f). Hence, MDDC must have formed processes before particles were applied, which is consistent with the findings in co-cultures with A549 cells (Figure 4a).

Immediately before and during particle exposition, a TEER around 500 {Omega}cm2 was measured in 16HBE140 co-cultures and in A549 co-cultures around 200 {Omega}cm2 (Figure 4a). TEER values measured before particle exposition were compared with values measured after particle exposition. Interestingly, TEER persisted in both types of co-cultures with no significant deviation (P > 0.05) throughout and after particle exposure (Figure 5a). Confluent monolayers of 16HBE14o cells show extensive TJ belts (34). The penetration of these TJ belts by MDDC was visualized in a co-culture with 16HBE14o cells stained for the transmembrane TJ protein occludin (Figure 5b).


Figure 5
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  		Figure 5. Epithelial monolayer integrity of co-cultures. (a) MDDC formed processes that penetrated (arrowheads) through the TJ network (occludin labeling, purple) toward the "luminal space." (b) TEER of A549 monocultures and 16HBE14o monocultures and TEER of co-cultures before and after particle addition. Each value represents the mean ± SE of at least five individual inserts (three measurements per insert).

 
MDDC and MDM Build a Transepithelial Network
In additional experiments, monocultures of MDM were pre-incubated with 1-µm particles and the particle-filled MDM were added to co-cultures of MDDC and epithelial cells. MDDC not only directly sampled particles deposited on the epithelium but also interacted with particle-loaded MDM on top of the epithelium. MDDC pushed processes up between the epithelial cells and "embraced" particle-filled MDM (Figures 6a–6a''). Particles were concentrated inside these processes surrounding the MDM, which might be evidence for immediate particle uptake from MDM. Particles, which were already transported across the epithelium, were found in processes of the same MDDC underneath the monolayer (Figure 6a''). Underneath the insert membrane MDDC were observed building a network with cell–cell contacts via extended processes (Figure 6b). Shortly after particle exposition, particles could be found near such cell–cell contacts, which could be an indication for a particle transfer between cytoplasmic processes of different MDDC (Figure 6b).


Figure 6
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  		Figure 6. Interplay of MDDC with MDM and other MDDC after particle exposure. (a–a'') Four hours after particle exposure, processes of MDDC (yellow) from below the epithelium (Ep) (actin, red) made contacts with a particle-filled MDM (blue) on top of the epithelium. Particles were found inside the MDDC embracing the MDM (1, arrows), and below the epithelium (2 and 3, arrows). Insets I in a and a'' show the MDDC–MDM interaction from a different angle. (b) Twenty-four hours after particle exposure, underneath the insert membrane MDDC formed a network (left panel) with numerous cell–cell contacts (asterisks). Particles were found inside MDDC (right panel, arrows) and near cell–cell contacts of different MDDC (inset I, arrows). a: maximum intensity projection of the xy-viewing direction (particles: white). a', a'', and b: surface rendering (particles: red). Bar is 20 µm.

 

   DISCUSSION
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
In spite of a series of structural and functional barriers, which are supposed to protect the respiratory system from inhaled particulate antigen (1), respiratory diseases are frequent and increasing (15–17). More attention has been directed toward elucidating how and when the antigens evade these barriers. Dendritic cells, as the most competent antigen-presenting cells, located in a network in and beneath the lung epithelium (22), play an important part in antigenic and pathogenic responses (29, 37, 38). In their immature stage they show a high capacity for antigen uptake but a low ability to stimulate T cells. After activation, dendritic cells can no longer take up antigens, but have a high stimulating capacity (22, 28). Our in vitro model showed a network of MDDC underneath the insert membrane similar to the network of dendritic cells already shown in vivo in tangential sections of the rat tracheal epithelium (26, 39). Furthermore, the cell density of MDDC around 400 cells/mm2 within our co-culture models was comparable with the steady-state cell density of 500–750 cells/mm2 reported from in vivo studies (22). Interestingly, in the absence of particles the arrangement of MDDC within the co-culture was the same. Even with no deposited particles at the apical side of the epithelium, MDDC extended processes between the epithelial cells into the "luminal space." In samples fixed within 10 min after particle exposition, particles could already be found inside these processes and even inside MDDC located underneath the insert membrane. Thus, the particle uptake was rapid, followed by immediate transport inside the processes across the epithelium. Particles could also be found near cell–cell contacts of MDDC underneath the insert membrane, indicating a possible particle exchange between MDDC in the network. Furthermore, there was a small fraction of MDDC in both co-culture models that did not remain stationary in the particle-exposed cultures but migrated completely to the top of the epithelium to collect particles from its surface. In A549 co-cultures significantly more MDDC were found on top of the epithelium 10 min and 24 h after particle exposure. An in vivo study with rats showed an increased migration of dendritic cells into and out of the lumen of the lung, which had been exposed to 500-nm latex beads after the depletion of alveolar macrophages using clodronate-loaded liposome (40). The authors suggested that these observations were due to suppressive effects of the macrophages on the dendritic cell activity and maturation, for example the secretion of nitric oxide (40). Our study, however, reveals the importance of the integrity of the epithelial monolayer on MDDC activity by demonstrating a higher migration of MDDC across the A549 monolayer expressing low TEER in contrast to the lower MDDC migration across the 16HBE14o monolayer expressing higher TEER, while the number of MDM in both co-cultures stayed constant. Similar results were found in a human in vivo study in which the epithelial barrier function was affected in allergic rhinitis and dendritic cells penetrated beyond the epithelium of the nasal mucosa (25). In our model, the mechanism that is responsible for the migration of MDDC is still unknown. Whether cytokines produced either by epithelial cells or MDM on the apical side play a role needs further investigation. The fact that, 4 h after particle exposition, no difference in MDDC migration could be found among the two culture systems but less MDDC could be observed before and after that time point in the 16HBE14o co-cultures compared to the A549 co-cultures, could point to a trend of MDDC in 16HBE14o co-cultures "lurking" in or beneath the epithelium before particle exposition. After particle exposition, MDDC may migrate to the top of the epithelium to sample deposited particles and may finally move back through the epithelium. This would correspond to in vivo findings of the migration of dendritic cells from the internal lung surface to the draining lymph nodes that has been reported (41). However, within both co-culture models no difference in MID could be found at any time after particle exposition, showing no accumulation of MDDC at any location within the three-dimensional models over 24 h.

In our model, MDDC acted as gatekeepers to sample noninvading 1-µm particles deposited on an epithelial monolayer and to transport them across the epithelium. These observations support the role of dendritic cells as very active sentinels in the immune surveillance of the airways (39) on the luminal surface and within the airway wall.

Alveolar macrophages as professional phagocytes account for approximately 95% of airspace leukocytes and are therefore considered as the sentinel phagocytic cells (42). In our study an obvious clearance of particles from the "luminal surface" of the epithelium was detected within 24 h, and most of the MDM were filled with particles 24 h after particle exposition. The evaluation of the PI of MDM confirmed the microscopic observation, since around 80% of all MDM contained particles in both co-culture models 24 h after particle exposition. The PI of MDDC, however, remained between 30 and 50% 24 h after particle exposure. These results are consistent with previous findings using in vitro monocultures of MDM or MDDC (36, 43). The visible clearance of particles was therefore mainly due to the highly phagocytic nature of macrophages, but apparently MDDC were joining MDM in their cleaning up task. Particularly in A549 co-cultures, significantly more MDDC were involved in particle uptake at the beginning of particle exposition compared with MDDC in co-culture with 16HBE14o cells, which showed a markedly lower TEER than A549 co-cultures. Furthermore, during exactly the same time PI of MDM in A549 co-cultures was significantly lower compared with the 16HBE14o co-cultures. These results clearly show the influence of MDDC on the PI of MDM within our culture models and demonstrate a close interplay between two cell types of the respiratory defense system. In analogy to the discussion of the migration of MDDC across the epithelium, we postulate that the higher PI of MDDC in A549 co-cultures compared with 16HBe14o co-cultures was due to the lower membrane integrity that facilitated the access of MDDC to the deposited particles on top of the epithelium within the A549 co-cultures. For a density of around 200 MDM/mm2 found in our co-cultures an epithelial surface area of 5,000 µm2 per MDM was calculated. Allometric analysis in the human lung revealed a surface area around 18,000 µm2 per macrophage in the alveolar regions (44). Thus, the density of MDM in our in vitro model was about 3.5 times higher compared with the steady-state in vivo situation in the human alveolar region. In vivo (45, 46) and in vitro (46) studies showed that alveolar macrophages could inhibit the maturation of the dendritic cell antigen-presenting functions. In both of our in vitro systems we observed a rather high involvement of MDDC in particle uptake, namely between 30 and 50%, 24 h after particle exposition. Thus the high density of MDM observed in our system might inhibit the maturation of MDDC and keep them in an immature state, consequently with high phagocytic activity (21). However, to clearly answer this question MHC class II expression in MDDC should be measured. Interestingly, in our studies using the co-cultures only few particles were found inside and none were found between epithelial cells of both cell lines. In another study with A549 monocultures we found nearly 40% of the total particle number inside epithelial cells 24 h after particle exposition (32). In the absence of competitive phagocytic cells, the epithelial cells appeared to have taken over their task of removing particles from the surface.

However, MDM and MDDC not only interacted indirectly during particle uptake, we also found evidence of a direct interplay between the two cell types. MDM were found to extend processes between the epithelial cells to the basal side where most of the MDDC were observed. This might be an indication for MDM seeking an interaction with MDDC underneath the epithelium. Even MDM without ingested particles were found to have processes toward the base of the epithelium (data not shown). Formation of processes was therefore either not dependent on particle uptake or particles might have already been passed to other cells, for example MDDC.

Clear evidence of MDDC–MDM interaction was found on top of the epithelium, where processes of MDDC which were pushed up between the epithelial cells embraced particle filled MDM. These processes contained many particles near cell–cell contacts of MDM and MDDC, and some particles had already been transported across the epithelium within MDDC. Although MDM and MDDC both are derived from circulating blood monocytes, macrophages have only weak antigen-presenting capabilities (47–50), but are twice as phagocytic as immature MDDC in vitro (36). A transfer of particulate antigen from macrophage to dendritic cell therefore makes sense, because macrophages may just be used as antigen carriers to transfer an immune response to dendritic cells. Other studies have described a transfer of macrophage-derived antigens to dendritic cells (47, 49) and antigen transfer in co-cultures of macrophages, dendritic cells, and T- and B-cells with dendritic cells (48). However, none of these studies considers the cellular interplay during antigen uptake and translocation as we did with our three-dimensional model. The immune response can be amplified not only through cell–cell contact but also through the release of cytokines and immunogens into the cellular environment. It has been reported that follicular dendritic cells stimulate B-cells in the lymph nodes by the release of iccosomes (51), or that dendritic cells stimulate T-cells by the release of exosomes (52). If MDDC of our cell culture system can stimulate other immune cells by the release of immunogens like iccosomes or exosomes is subject of current investigations.

Although the TJ belt was penetrated by processes of MDDC and MDM to transfer particles across the epithelium and MDDC migrated through the epithelium, the monolayer integrity of both epithelial cell lines was not affected at any time point before and after particle exposition. The persistence of TEER during the exposition experiments indicated that the apical TJ belt of the epithelium never was disrupted. From studies with cultured human gut epithelial cells it is known that dendritic cells express TJ proteins to form TJ-like complexes with epithelial cells in order to preserve the epithelial integrity (23, 53). Furthermore a population of dendritic cells residing in the mucosa of mouse airways was recently found to express the TJ proteins Claudin-1, Claudin-2, and ZO-2 (54). Cell processes of MDDC in our model may also make temporal TJs with the epithelial cells. Evidence for this comes with the TEER, which never dropped during all the experiments. Nevertheless, further investigations with our model are needed to study TJ protein expression in MDDC and especially in MDM, as they were also found to extend processes through the epithelial TJs.

Using LSM combined with digital image restoration, we visualized two proposed mechanisms of transport of particulate antigen across the epithelial barrier (23–25, 27–29, 53) in a human cellular model of the airway epithelium (Figure 7): (1) uptake of particles by extended cytoplasmic processes of MDDC across the epithelium, and (2) transmigration of complete MDDC through the epithelium to capture particles. Moreover, we found evidence for three further mechanisms of particle translocation to MDDC (Figure 7): (3) particle exchange between MDDC below the epithelium; (4) particle transfer from MDM to MDDC via cell–cell contacts between MDDC and particle filled MDM on the "luminal side" of the epithelium; and (5) transfer of particles through processes of MDM, which may interact with MDDC in or underneath the epithelium.


Figure 7
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  		Figure 7. Transepithelial interactions. Cellular interplay of epithelial cells, MDM, and MDDC after particle exposure in a cell culture model of the human airway wall; epithelial cells (red), MDM (blue), MDDC (yellow), and particles (white). 1, extension of cytoplasmic processes by MDDC; 2, transmigration of complete MDDC; 3, particle transfer from MDDC to MDDC in or at the base of epithelium; 4, particle transfer; 5, MDM extending processes in the epithelium to the basal side of the epithelium.

 
Despite the fact that cultured MDDC might not reflect the exact nature of an endogenous pulmonary dendritic cell population in terms of phenotype, activation level, and antigen uptake capacity, our in vitro model offers a system for the study of the interplay between dendritic cells, macrophages, and epithelial cells. We conclude that dendritic cells and macrophages compete, but to a greater degree collaborate as sentinels against particulate antigens in a transepithelial network. These two cell types are the main players in the defense against particulate antigens. With an extended fine transepithelial cytoplasmic network, they appear to handle in a very effective manner a transcellular transepithelial transport of antigens from the "luminal side" to the base of the membrane. With our cell culture model we will further investigate particle–cell and cell–cell interactions, in order to study the fate of particles after their transepithelial transportation.


   Acknowledgments
 
The authors thank Prof. Patrick Holt for stimulating discussions. They are grateful to A. Luginbuehl, B. Tschirren, and S. Frank for technical assistance. They are indebted to Dr. Gruenert (University of California, San Francisco) for providing the 16HBE14o cell line.


   Footnotes
 
* These authors contributed equally to this work. Back

This work was supported by the Swiss National Science Foundation (Nr. 32-65352.01), the Federal Office for the Environment, and the Silva Casa Foundation.

Originally Published in Press as DOI: 10.1165/rcmb.2006-0234OC on February 1, 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 29, 2006

Accepted in final form January 24, 2007


   References
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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