Am J Physiol Lung Cell Mol Physiol 302: L226 –L237, 2012.
First published November 18, 2011; doi:10.1152/ajplung.00059.2011.
Dendritic cell functional properties in a three-dimensional tissue model
of human lung mucosa
Anh Thu Nguyen Hoang,1 Puran Chen,1 Julius Juarez,1 Patty Sachamitr,2 Bo Billing,3 Lidija Bosnjak,1
Barbro Dahlén,3 Mark Coles,2 and Mattias Svensson1
1
Center for Infectious Medicine at the Department of Medicine, Karolinska Institutet, and 3Division of Respiratory Medicine
and Allergy at Karolinska University Hospital, Huddinge, and Center for Allergy Research, Karolinska Institutet, Stockholm,
Sweden; 2Centre for Immunology and Infection, at Hull York Medical School and Department of Biology, University of York,
York, United Kingdom
Submitted 25 February 2011; accepted in final form 12 November 2011
3D organotypic model; lung mucosal tissue; epithelial cells; fibroblasts; immune regulation; chemokines
TISSUE HOMEOSTASIS IS A DYNAMIC process dependent on the
cooperation between immune cells, tissue specific-nonimmune
cells, and extracellular matrix components (23, 34, 40). Although technologies have been developed to study cell-cell and
cell-matrix interactions during homeostasis and inflammation
in three-dimensional (3D) environments in vivo, these approaches are often limited to animal models (11, 28). Therefore, there is a need to develop a new cell-based in vitro human
tissue model relevant to the understanding of mechanisms
regulating homeostasis and inflammatory responses in the
lung. In particular, such approaches can contribute toward
increased understanding of the dynamic interactions between
human nonimmune and immune cells within specific tissues.
Address for reprint requests and other correspondence: M. Svensson, Center
for Infectious Medicine, F59, Dept. of Medicine, Karolinska Institutet, Karolinska Univ. Hospital, Huddinge, 141 86 Stockholm, Sweden (e-mail:
[email protected]).
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Human tissue models can also capture important aspects of
tissue-associated responses to infections because many important human pathogens [e.g., Mycobacterium tuberculosis (M.
tuberculosis), group A streptococcus, and Staphylococcus aureus] induce species specific responses (2, 25).
Several approaches have been used to study immune responses of human lung mucosa to external stimuli in vitro,
including monolayers of epithelial cells isolated from lung
tissue bronchoscopies (37). Epithelial cells isolated from bronchoscopy are terminally differentiated, short-lived cells that
generally have lower metabolic capacity than actively growing
cells in vivo or in vitro (14). Although, primary bronchial
epithelial cells can develop tight junctions and adherence
junctions as well as deposit extracellular matrix proteins found
in functional mucosal barriers, they may have a variable
constitutive and inducible expression of proteins, depending on
the donor as previously reported (42). These monolayer systems are additionally limited by the lack of polarized cell
phenotype and the lack of a large number of cell-cell contacts,
which will affect their function and response to external stimuli
(31). Instead, the complexity of two-cell layer-based systems
are advantageous as demonstrated when studying, for example,
early events in M. tuberculosis infection (2).
More recently, protocols describing the setup of bronchial
3D mucosal tissue models that better recapitulate human lung
mucosa have been developed (6). This includes models with a
physiologically relevant fibroblast matrix layer and epithelial
differentiation into ciliated, mucus-secreting cells (6, 9). Under
such conditions, fibroblasts proliferate slowly, and their extracellular matrix is likely to provide better conditions for growth
and differentiation of lung epithelial cells in vitro compared
with artificial gels or membranes, possibly via the release of
growth and survival factors (5, 15). The fibroblast component
is, not only critical in promoting survival, remodeling, and
deposition of matrix components, but also ensures the resemblance to the human lung mucosa and submucosa (4, 5).
Fibroblasts actively interact with the adjacent epithelial layer
and have a key role in inflammation and repair of lung tissue
(36). What has only recently been recognized are the cellular
interactions between epithelial cells, fibroblasts, and immune
cells, such as dendritic cells (DC), in many aspects of tissue
homeostasis and inflammation. Thus, in vitro 3D models combining DC with tissue-specific cells provides a useful tool to
study mechanisms involved in local inflammatory processes
previously not achievable, and will have a great potential in
replacing, in some cases, experiments performed on animals.
1040-0605/12 Copyright © 2012 the American Physiological Society
http://www.ajplung.org
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Nguyen Hoang AT, Chen P, Juarez J, Sachamitr P, Billing B,
Bosnjak L, Dahlén B, Coles M, Svensson M. Dendritic cell functional
properties in a three-dimensional tissue model of human lung mucosa.
Am J Physiol Lung Cell Mol Physiol 302: L226 –L237, 2012. First
published November 18, 2011; doi:10.1152/ajplung.00059.2011.—In
lung tissue, dendritic cells (DC) are found in close association with the
epithelial cell layer, and there is evidence of DC regulation by the
epithelium; that epithelial dysfunction leads to overzealous immune cell
activation. However, dissecting basic mechanisms of DC interactions
with epithelial cells in human tissue is difficult. Here, we describe a
method to generate a three-dimensional organotypic model of the human
airway mucosa in which we have implanted human DC. The model
recapitulates key anatomical and functional features of lung mucosal
tissue, including a stratified epithelial cell layer, deposition of extracellular matrix proteins, and the production of tight junction and adherence
junction proteins. Labeling of fixed tissue model sections and imaging of
live tissue models also revealed that DC distribute in close association
with the epithelial layer. As functional properties of DC may be affected
by the local tissue microenvironment, this system provides a tool to study
human DC function associated with lung mucosal tissue. As an example,
we report that the lung tissue model regulates the capacity of DC to
produce the chemokines CCL17, CCL18, and CCL22, leading to enhanced CCL18 expression and reduced CCL17 and CCL22 expression.
This novel tissue model thus provides a tool well suited for a wide range
of studies, including those on the regulation of DC functional properties
within the local tissue microenvironment during homeostasis and inflammatory reactions.
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DC REGULATION BY LUNG TISSUE-SPECIFIC CELLS
proteins and the formation of tight junction and adherence
junction proteins. Analyses of fixed-tissue models also revealed that DC distribute in close association with the epithelial layer, and this was confirmed also in live tissue models
using multiphoton microscopy analysis. In addition, we found
that the lung tissue microenvironment affects DC function and
that the chemokines CCL17, CCL18, and CCL22 are differentially regulated in DC within the 3D model. Thus this system
provides a new platform to study human DC functional properties associated to lung mucosal tissue, previously not achievable.
MATERIALS AND METHODS
Cell lines. MRC-5 is a human lung fibroblast cell line (American
Type Culture Collection, Manassas, VA) derived from normal lung
tissue of a 14-wk-old male fetus. MRC-5 were maintained in complete
DMEM (Invitrogen, Carlsbad, CA) with 1 mM sodium pyruvate, 2
mM L-glutamine, 100 U/ml penicillin, 100 ␮g/ml streptomycin, 10
mM HEPES, 0.1 mM nonessential amino acids, and 10% heatinactivated FBS (all from Invitrogen). The MRC-5 fibroblasts were
thawed and expanded on tissue-culture flasks in complete DMEM
until 70 – 80% confluent (⬃7–9 days) before use in the model. The
cells were used at passages 24 –26 in the model.
The cell line 16HBE (a gift from Dr. Dieter Gruenert, Mt. Zion
Cancer Center, University of California, San Francisco, CA) is an
immortalized human bronchial epithelial cell line developed by transformation of human surface epithelial cell from 1-yr-old male with
SV40 large T-antigen. 16HBE were cultured in fibronectin/collagencoated flasks (7) and maintained in complete MEM (Sigma-Aldrich,
St. Louis, MO) supplemented with Earle=s salts, 0.292 g/l L-glu-
Fig. 1. Lung tissue model setup and histological analysis of the model. A: schematic
drawing of a previously established human
lung tissue model combining a fibroblast
collagen layer with stratified epithelial cells.
B: schematic drawing of our newly developed human lung tissue model composed of
MRC-5 fibroblasts and stratified 16HBE epithelial cell layer in combination with monocyte-derived dendritic cells (DC). C: time
frame and different stages involved in the
lung tissue model setup. D: hematoxylin and
eosin staining of an 8-␮m cryosection of the
lung tissue model is shown (original magnification ⫻200). Higher magnifications show
a structure equivalent to a basement membrane (BM) at the boundary between epithelial cells and the underlying fibroblast matrix
layer (inset: original magnification ⫻1,000).
E: hematoxylin staining of an 8-␮m cryosection of control lung tissue, is shown (original
magnification ⫻200). The bronchial biopsy
was taken proximally in the right upper lobe
bronchus.
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DC belong to a heterogeneous population of widely distributed immune cells that play a central role in initiation and
regulation of immune responses (1). Immune responses to
pathogens are initiated and orchestrated by DC located in
peripheral tissues, including the skin and mucosa. DC orchestrate immune responses locally by producing cytokines and
chemokines that are important in the activation and recruitment
of other inflammatory cells (22). Within lung tissue, DC
mainly associate with the epithelial layer, and the interactions
with the epithelium likely influence the phenotype and function
of DC, including their ability to orchestrate immune responses
locally (12, 18). There are also data supporting that epithelial
cell dysfunction leads to overzealous immune activation (17).
However, dissecting how tissue-specific nonimmune cells and
DC cooperate in coordinating tissue homeostasis and inflammation in human tissue has multiple practical problems. Although 3D lung tissue models that resemble “real” tissue have
been engineered to include a fibroblast extracellular matrix and
differentiating epithelial cells (Fig. 1A), few, however, are
utilizing the combination of fibroblasts, epithelial cells, and
immune cells, such as DC that can be imaged in 4D (x, y, z,
time, Fig. 1B).
In this study, therefore, we developed a standardized human
3D organotypic lung model using the “normal” bronchial
epithelial cell line 16HBE14o⫺ (16HBE) immortalized with
the SV40 large T antigen, the MRC-5 fibroblasts derived from
fetus lung tissue, and human monocyte-derived DC. The model
recapitulates key anatomical and functional features of lung
mucosal tissue, including deposition of extracellular matrix
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DC REGULATION BY LUNG TISSUE-SPECIFIC CELLS
ferred to ⫺80°C until required. Eight-micron cryosections were cut
onto diagnostic microscope glass slides (Thermo Scientific, Waltham,
MA) using a MICROM cryostat HM 560 MV (Carl Zeiss, Jena,
Germany) and fixed in 2% freshly prepared formaldehyde in PBS for
15 min at room temperature or in ice-cold acetone for 10 min at
⫺20°C. For hematoxylin and eosin staining, the sections were stained
for 15 s in Mayes Hematoxylin and counterstained for 2 min in eosin.
For immunohistochemistry, 8-␮m frozen sections were blocked
with 10% FBS in balanced salt solution (BSS) with 0.1% saponin for
30 min at room temperature, followed by additional blocking with 2%
H2O2 in BSS-saponin and an avidin biotin blocking reagent (Vector
Laboratories, Burlingame, CA). Primary monoclonal rat anti-human
leukocyte antigen (HLA)-DR (1.25 ␮g/ml, clone YE2/36-HLK; AbD
Serotec, Dusseldorf, Germany) antibody was diluted in BSS solution
containing saponin and incubated overnight at room temperature as
previously described (27). After incubation, tissue sections were
washed and blocked with 1% normal rabbit serum in BSS-saponin
before addition of biotinylated rabbit anti-rat IgG (4.2 ␮g/ml; Dako,
Glostrup, Denmark) diluted in 1% normal rabbit serum in BSSsaponin. After wash avidin-peroxidase solution was added (Vectastain-Elite; Vector Laboratories), and the color reaction developed
by the addition of 3,3-diaminobenzidine (Vector Laboratories) followed by counterstaining with hematoxylin. Tissue sections were
visualized using a Leica DMR microscope (Wetzlar, Germany).
Imaging and quantification of positive immunostaining were performed using acquired computerized image analysis by transferring
digital images of the stained tissue samples taken by a DMR-X microscope (Leica) to a computerized Quantimet 5501W image analyzer
(Leica) (3). Single-positive stained cells were quantified in 25 high-power
fields, and protein expression was determined as the percent positive area
of the total relevant cell area using a Qwin 550 software program (Leica
Imaging Systems). The total cell area was defined as the nucleated and
cytoplasmic area within the tissue. Tissue sections stained with secondary
antibodies only were used as negative controls.
Alcian blue staining. Eight-micron-thick frozen tissue sections
were hydrated in distilled water and stained in Alcian blue solution
(pH 2.5, Sigma Aldrich) for 30 min at room temperature. The sections
were washed for 2 min in tap water and then rinsed in distilled water.
After that, sections were counterstained in nuclear Fast Red solution
(Sigma Aldrich) for 5 min and were washed in tap water for 1 min. The
sections were dehydrated through 95% alcohol and twice in absolute
alcohol for 3 min each. The sections were cleared in xylene and mounted
in DPX mountant (VWR International, Radnor, PA). Mucins and mucosubstances were thus stained in blue, nuclei in pink to red, and cytoplasm
in pale pink.
Immunofluorescence analysis. Immunofluorescent labeling of
8-␮m frozen sections was performed with the following primary
antibodies: mouse anti-type IV collagen (13.5 ␮g/ml, clone COL-94;
Abcam, Cambridge, UK), rabbit anti-tropoelastin (1:200, polyclonal;
Elastin Products, Owensville, MO), mouse anti-laminin-5 (␣3) (2.5
␮g/ml, clone P3H9 –2; R&D Systems, Minneapolis, MN), mouse
anti-vimentin (1:100, clone VIM3B4; Novocastra Laboratories, Newcastle, UK), rabbit anti-claudin I (1 ␮g/ml, polyclonal; Abcam),
mouse anti-occludin (2.5 ␮g/ml, clone OC-3F10; Invitrogen), mouse
anti-E-cadherin (2 ␮g/ml, clone HECD-1; Invitrogen), rat antiHLA-DR (2.5 ␮g/ml, clone YE2/36-HLK; AbD Serotec), mouse
anti-DC-specific ICAM-grabbing nonintegrin (SIGN) (0.25 ␮g/ml,
clone 120507; R&D Systems), and mouse anti-CD11c (1.25 ␮g/ml,
clone B-ly6; BD Pharmingen, San Diego, CA). Sections were washed
in PBS containing 0.2% saponin and blocked with 1.5% normal goat
serum (NGS) in 0.2% saponin in PBS for 45 min at room temperature.
Sections were incubated with primary antibodies (see above) diluted
in PBS containing 1.5% NGS and 0.2% saponin for 45 min at room
temperature and then washed three times in PBS with 0.2% saponin.
Specific staining was detected by the following secondary antibodies:
Alexa 488-conjugated goat anti-rat IgG, Alexa 488-conjugated goat
anti-mouse IgG, Alexa 555-conjugated goat anti-mouse IgG2b, Alexa
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tamine, 2.2 g/l sodium bicarbonate, 100 U/ml penicillin, 100 ␮g/ml
streptomycin, and 10% heat-inactivated FBS (Invitrogen) in 5% CO2
at 37°C. The cells were used at passage 74. 16HBE were then
maintained in complete DMEM when cultured together with the
fibroblasts and DC in the model.
Generation and conditioning of monocyte-derived DC. Monocytes
were isolated from buffy-coated blood using human monocyte enrichment cocktail RosetteSep (StemCell, Grenoble, France). The cells
were separated by ficoll gradient separation, washed twice in PBS,
and subsequently cultured in complete DMEM supplemented with
12.5 ng/ml IL-4 (R&D Systems Minneapolis, MN) and 50 ng/ml
colony stimulating factor (CSF)-2 (Peprotech, Rocky Hill, NJ) for 6
days. Fresh media including concentration of cytokines (25 ng/ml
IL-4, 100 ng/ml CSF-2) was provided on day 3 of culture. DC
generated from monocytes were routinely positive for CD1a and
negative for CD14 (⬎ 90%). For the conditioning experiments, DC (5
⫻ 104 cells) from day 6 were conditioned by replacing the original
medium with 500 ␮l of culture supernatants from the lung tissue
model composed of only fibroblasts and epithelial cells, or with
supernatants from either fibroblasts or air-exposed epithelial cells, or
with supernatants from a mixed monolayer of both fibroblasts and
epithelial cells. The DC were conditioned for 24 h and then extracted
for RNA, and supernatants were collected for protein measurement
using ELISA. Ethical permission for the use of human tissue and cells
was obtained from the regional ethics committee at Karolinska Institutet, Stockholm, Sweden, and where needed informed consent has
been obtained.
Organotypic model preparation. The models were generated on
3.0-␮m Transwell inserts (Becton Dickinson, Franklin Lakes, NJ) in
six-well plates based on a protocol for human oral mucosal models
(9). The procedure of establishing model tissue takes ⬃4 –5 wk (Fig.
1C) and includes cell expansion, culturing of the cells in the model,
and air exposure (AE) of the tissue model. To set up the model, the
inner chamber of the six-well inserts were coated with a solution of
bovine type I collagen (1.1 mg/ml; Organogenesis, Canton, MA) in
DMEM and incubated for 30 min at 37°C. A suspension of MRC-5
fibroblasts at 2.3 ⫻ 105 cells/ml diluted in 1.1 mg/ml of collagen
DMEM mixture was added onto the precoated collagen layer and
incubated for 2 h in 5% CO2 at 37°C. Following polymerization, 2 ml
of complete DMEM was added to the outer chamber, and the cultures
were incubated for an additional 24 h. The following day, culture
media was removed from the outer and the inner chamber. Then 2 ml
of fresh complete DMEM was added to both chambers. To allow
remodeling of the stroma/matrix layer, cultures were incubated for 7
days with the medium being replaced every second day.
After 7 days, the culture media was removed from the outer and
inner chambers followed by the addition of 1.5 ml of fresh complete
DMEM to the outer chamber. Then 2 ⫻ 105 monocyte-derived DC in
50 ␮l of complete DMEM were added to the fibroblast collagen
matrix and incubated for 2 h in 5% CO2 at 37°C. After incubation 1.5
ml of culture media was gently added into the insert, and the culture
was incubated for an additional 24 h in 5% CO2 at 37°C before
seeding of the epithelial cells. Epithelial cells were seeded by adding
5 ⫻ 104 epithelial cells in 50 ␮l complete DMEM to each insert.
Submerged cultures were incubated for an additional 3 days in 5%
CO2 at 37°C to allow the epithelial cells to form a confluent monolayer on the collagen gel. After 3 days, the tissue models were air
exposed by removing the medium from the insert and reducing the
volume to 1.5 ml in the outer chamber. Air-exposed tissue models were
incubated for up to 10 days in 5% CO2 at 37°C, and the culture media in
the outer chamber was changed every second day. At days 5 to 10, tissue
models were used for immunohistological immunofluorescence and livecell imaging analyses, as well as DC functional analyses.
Histological and immunohistological analyses. Lung tissue model
for cryosectioning was prepared by embedding the tissue in optimal
cutting temperature compound (Sakura Finetek Europe, Zoeterwoude,
Netherlands) and then slowly frozen in ⫺20°C for 24 h and trans-
DC REGULATION BY LUNG TISSUE-SPECIFIC CELLS
was performed at 872–930-nm wavelengths using a C-Apochromat
⫻40 1.1 NA water dipping objective.
Real-time qRT-PCR analysis. Total RNA was extracted from DC
and from the lung tissue model by using a Ribopure kit (Applied
Biosystems, Foster City, CA) following the manufacturer’s protocol.
To compare chemokine expression between the 3D model containing
DC and the control DC, we used 1.5 ⫻ 105 DC (similar numbers of
cells that was implanted in each 3D model) cultured in 1.5 ml of
complete DMEM without CSF-2 and IL-4 for 24 h before analysis.
Similar conditions for control DC and DC implanted in the 3D model
were obtained by lysing control DC in TRIzol together with one 3D
model (fibroblasts and epithelial cells only) before RNA extraction.
RNA was converted into cDNA by using a cDNA reverse transcription kit (Applied Biosystems) according to the manufacturer’s instructions. The primers and probes for HLA-DR, DC-SIGN, CD11c,
CSF-2, IL-4, IL-1␤, TNF, IL-10, IL-13, transforming growth factor
(TGF)-␤, CX3CL1, CXCL8, CCL17, CCL18, CCL22 and ubiquitin C
were purchased as Predeveloped TaqMan Gene Expression Assays
(Applied Biosystems). Ubiquitin C served as an endogenous control to
normalize the amount of sample cDNA. Amplification and quantification of cDNA was performed as previously described (27). Relative
amounts of the two chemokines were calculated using the comparative threshold cycle (CT) method. The threshold correlates to the cycle
number where there is sufficient amplified product to give a detectable
reading, and, if the threshold is not attained after 35– 40 cycles,
the mRNA is considered undetectable. The data are presented as
relative expression of chemokine mRNA in untreated DC conditioned
with supernatant compared with the chemokine mRNA in control DC.
The data are also presented as relative expression of mRNA in the
lung tissue model containing DC compared with control DC (mixed
1:1 with RNA extracted from a 3D model without DC) and 3D model
composed of epithelial cells and fibroblasts only.
Chemokine measurement. Supernatants from control DC and tissue
model were collected for protein measurement. A sandwich ELISA
construction kit to detect CCL18 (R&D Systems), CCL17, and
CCL22 (Antigenix, Huntington, NY) was used according to the
manufacturer’s protocols. CCL18 was detected using 1.0 ␮g/ml capture of antibody and 200 ng/ml of detection antibody. CCL17 was
detected using 1.0 ␮g/ml capture of antibody and 0.2 ␮g/ml detection
antibody. CCL22 was detected using 1.0 ␮g/ml of capture antibody
and 0.25 ␮g/ml detection antibody. Assays were developed by adding
streptavidin-peroxidase and 3,3,5,5-tetramethyl-benzidine as the substrate. Absorbance was read at 450 nm using a Microplate Manager 6
reader (Bio-Rad, Hemel Hempstead, UK). Recombinant CCL18 (1
ng/ml), CCL17 (10 ng/ml) and CCL22 (10 ng/ml) were used as
standards, and the concentration of chemokines in the test samples
was calculated using the linear part of a “four-parameter fit” standard
curve run in parallel with the samples.
Statistical analysis. Data are presented as means ⫾ SD for the
indicated number of experiments. All analyses are based on three or
more separate experiments performed with monocyte-derived DC
from different donors. The data were analyzed with use of the
GraphPad Prism v.5 software (GraphPad, San Diego, CA) using
one-way ANOVA with Bonferroni multiple-comparison test, and
differences between groups were determined to be statistically significant at P ⬍ 0.05.
RESULTS
Characterization of the lung tissue model. To reveal pathways by which human DC and tissue-specific nonimmune cells
cooperate to orchestrate lung tissue physiology and immune
responses at the mucosal surface, we sought to establish a 3D
organotypic lung model combining human DC with two distinct cellular compartments: a fibroblast/matrix layer and an
epithelial cell layer. The lung tissue model was cultured on a
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488-conjugated goat anti-mouse IgG1, Alexa 594-conjugated goat
anti-rabbit IgG (all from Molecular Probes, Invitrogen). The sections
were incubated with secondary antibodies diluted to 3.3 ␮g/ml for
Alexa 488 and 4 ␮g/ml for Alexa 555 and 594 in PBS containing
1.5% NGS and 0.2% saponin for 30 min at room temperature.
Sections were mounted in SlowFade Gold antifade reagent with
4,6-diamidino-2-phenylindole (DAPI) (Molecular Probes, Invitrogen)
and visualized using a Leica TCS SP2 confocal microscope (Leica).
Quantification of DC numbers over time was performed in live
tissue models by imaging the models at days 1, 3, 5, and 8 using a
confocal microscope. To enable DC visualization in live tissue, DC
were labeled with the far-red cell tracker, dimethyl dodecylamine
oxide-syccinimidyl (Molecular Probes, Invitrogen), before implantation into the models. Day 6 monocyte-derived DC were washed twice
with PBS, resuspended at 106 cells/ml of PBS, and labeled with 5 ␮M
of the cell tracker at room temperature for 15 min. Cells were washed
twice with PBS after being labeled and resuspended to 2 ⫻ 105
cells/50 ␮l of complete DMEM. The labeled DC were seeded in 50 ␮l
directly on top of the fibroblast collagen layer as described above.
After 24 h, epithelial cells were added to the models followed by the
quantification of DC (day 1). Additional 3D models with DC were air
exposed and cultured for DC quantification at days 3, 5, and 8. Tissue
models were visualized using a Nikon A1R confocal microscope
(Nikon Instruments, Amstelveen, The Netherlands), and the obtained
z-stack images were used for reconstruction of 3D projections. To
quantify DC in 3D microscopic projections, fluorescently labeled cells
within a defined field of each 3D projection were counted. A minimum of 250 cells was counted in each field, and, to exclude enumeration of dead cells, propidium iodide was added to the models before
imaging. Then the number of cells per defined field was multiplied
with a factor reflecting the total 3D tissue model area.
Generation of 16HBE cells expressing tdTomato. pCMV expression vector encoding the fluorescent tdTomato protein was a kind gift
from R. Tsien (San Diego, CA). Plasmids were purified using
HiSpeed MidiPrep kit (Qiagen, Crawley, UK) according to manufacturer’s instructions. Cells were cultured in 35-mm culture dishes
(MaTek, Ashland, MA), and, at 50 – 80% confluency, cells were
transfected utilizing 4 ␮g of pCMV-tdTomato plasmid diluted in 100
␮l OptiMEM and 14 ␮l Lipofectamine (Invitrogen) diluted in 100 ␮l
OptiMEM. Both solutions were mixed and incubated at room temperature for 30 min. Media was removed from 35-mm dish and
replaced with 600 ␮l OptiMEM and transfection mixture. After 6 h of
incubation at 5% CO2, 37°C, 1.2 ml of complete DMEM with 20%
FBS was added to the dish. The media was replaced with complete
DMEM the next morning. Transfection efficiencies were analyzed
using a CyAn flow cytometer (Beckman Coulter, Brea, CA). Cells
were selected in 400 ␮g G418 for 2 wk, and then the highest
expression fraction was isolated using high-speed MoFlo cell sorter
(Beckman Coulter). Nontransfected 16HBE cells were used to define
the background fluorescence and gating strategy. Postsort purity of
cells was analyzed using the CyAn ADP flow cytometer.
Live imaging of the tissue model using multiphoton microscopy.
The lung tissue model was generated for live imaging using fluorescent tdTomato 16HBE and carboxyfluorescein diacetate succinimidyl
ester (CFSE)-labeled monocyte-derived DC. Day 6 DC were washed
twice with PBS, resuspended at 106 cells/ml of PBS, and labeled with
5 ␮M of CFSE (Molecular Probes, Invitrogen) at room temperature
for 15 min. Cells were washed twice with PBS after labeling and
resuspended to 2 ⫻ 105 cells/50 ␮l of complete DMEM. The labeled
DC were seeded directly on top of the fibroblast collagen layer as
described above. After 7 days of AE, the fluorescent model was
removed from the membrane insert and placed onto a 35-mm glassbottom dish and imaged on a Zeiss LSM 510 NLO Meta Laser
Scanning Confocal Microscope (Carl Zeiss) with three detectors for
non-descanned detection (NDD) and a Coherent Chameleon Ultra
laser (Coherent, Santa Clara, CA) with a blacked out, temperature
controller chamber (Solent Scientific, Segensworth, UK). Imaging
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lagen matrix layer and not in the epithelial layer (Fig. 2C).
Together, this indicates that molecules forming essential structures for lung tissue function are produced and deposited in the
model.
The lung tissue model form tight and adherence junctions
and a mucus layer. One key function of mucosal barriers
resides in epithelial cell impermeability that is mediated by the
formation of tight and adherence junctions (19). To verify that
the epithelial cell layer of the lung tissue model has the
capacity to form tight junctions and adhesion junctions, we
analyzed the tissue sections for the presence of claudin I and
occludin, two proteins important for formation of mucosal
junction structures (19). As shown in Fig. 3A, epithelial cells in
the lung tissue model express claudin I. In addition, occludin
that interacts directly with claudins and actin (19) was detected
in the lung tissue model (Fig. 3B). In mucosal tissue, production of E-cadherin by epithelial cells is important for formation
and preservation of stratified epithelial barriers (19). Therefore,
the model was analyzed for the presence of E-cadherin, and
this revealed that E-cadherin is preferentially expressed by the
epithelial cells and not by the fibroblasts in the tissue model
(Fig. 3A and data not shown).
The production of mucus by the epithelium plays an important
role in the protection of epithelial barriers against infectious
agents and toxins that enter the respiratory system. To investigate
whether our epithelial cells secret mucus, we stained the model
with Alcian blue, a reagent that stains acid mucosubstances and
acetic mucins. This showed mucus production already after 5 days
of AE (Fig. 3C). Mucus production was also detected at 10 days
of AE (Fig. 3D). These data indicate that the tissue model has a
functional mucosal barrier and produce the tight junction proteins,
claudin I and occludin, the cell-cell adhesion molecule Ecadherin, and mucus. To conclude, we have created a human 3D
Fig. 2. The lung tissue model produces structural proteins important for lung tissue function. Immunofluorescence analyses of structural proteins essential for lung tissue function
were carried out on cryosections (8 ␮m) of
frozen lung tissue models. A and B: images
show distribution of collagen IV (green) and
tropoelastin (red) in the lung tissue model.
Higher magnification (original magnification
⫻630) shows localization of collagen IV and
tropoelastin fibrous structures (arrowed) at the
boundary between the stroma matrix and epithelial cell layer (B). C and D: images show
distribution of vimentin (C) and laminin-5 (D)
in the lung tissue model (original magnification ⫻200). 4,6-Diamidino-2-phenylindole
(DAPI) was used to reveal cell nuclei. All
results are representatives of at least 3 independent experiments. In each experiment, 6
tissue models were analyzed (original magnification ⫻200).
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permeable membrane for a total of 11–14 days, including
submersion in medium for 4 days and AE for 7–10 days. After
AE histological analysis of the tissue model revealed that the
epithelial cells had formed a stratified squamous structure on
top of the fibroblast matrix consistent with reports in similar
systems containing lung epithelial cells (Fig. 1D). Also, a
structure equivalent to a basement membrane was observed at
the boundary between epithelial cells and the underlying fibroblast matrix layer (Fig. 1D, inset). These data demonstrate that
our 3D organotypic lung model is fully developed and has a
structure that resembles normal lung mucosa tissue (Fig. 1E).
Extracellular matrix proteins are deposited in the lung tissue
model. Extracellular matrix proteins, such as collagens, elastin,
and laminins are essential for tissue to function properly. To
confirm that the tissue-specific cells in our model produce and
deposit important tissue components, immunofluorescent analysis of frozen tissue sections was performed. Sections were
labeled with antibodies against type IV collagen and tropoelastin, known to be important for formation of basement membranes. This revealed that both epithelial cells and fibroblasts
in the tissue model produce type IV collagen and tropoelastin
and that a continuous basement membrane is formed at the
boundary between epithelial cells and the underlying fibroblast
matrix layer (Fig. 2, A and B). Tissue sections were also
analyzed for the presence of laminin-5, an extracellular matrix
protein important in the initiation and maintenance of epithelial
cell anchorage to the underlying connective tissue layer (35).
Laminin-5 was found associated to epithelial layer as well as the
basement membrane at the boundary between fibroblasts and
epithelial cells (Fig. 2D). Immunofluorescence analysis also
confirmed that yet another protein, vimentin, that is important
for tissue fibroblast function was produced by the tissue model
(39). Vimentin staining was only found in the fibroblast col-
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lung tissue model that has basic structural and typical features of
a normal lung mucosa.
The lung tissue model supports DC survival independent of
exogenous growth factors. Next, we sought to confirm that DC
in our lung tissue model survived equally well in both the
absence and presence of CSF-2 and IL-4. Monocyte-derived
DC were added to the fibroblast/matrix layer before seeding
epithelial cells, and subsequently the models were cultured either
with or without addition of exogenous CSF-2 and IL-4 for 11
days. To visualize DC, lung tissue models were sectioned and
analyzed for the presence of HLA-DR⫹ cells using immunohistochemical analysis. The HLA-DR staining not only revealed that
DC have survived in the tissue model but also that the DC are
closely distributed to the epithelial layer consistent with reports of
DC location in the lung tissue (24). Acquired computerized image
analysis of HLA-DR immunostaining (Fig. 4, A–C) revealed that
DC in the tissue model survived equally well in the absence
and the presence of CSF-2 and IL-4 (Fig. 4D). These findings
demonstrate that the lung tissue model supports the survival of
DC independent of exogenous growth factors, CSF-2 and IL-4,
for at least 11 days. To investigate whether there is endogenous
expression of CSF-2 and IL-4 in the 3D lung model, we
performed real-time RT-PCR analysis of lung tissue model
without DC, DC cultured in medium only, and the lung tissue
model with DC (Fig. 4E). CSF-2 mRNA was abundantly
expressed, in all samples compared, and CSF-2 expression was
lower in DC (average Ct value ⫽ 27.1 ⫾ 0.8) and the 3D
model with DC (average Ct value ⫽ 28.9 ⫾ 1.4) compared
with the 3D model only (average Ct value ⫽ 26.7 ⫾ 0.5). In
contrast, IL-4 mRNA was undetectable in all samples compared. Recent studies have established CX3CL1/fractalkine,
the ligand for CX3CR1, as an important chemokine for DC
interaction with the epithelial layer in lung tissue. The
CX3CL1 mRNA was abundantly expressed in the lung tissue
model without DC (average Ct value ⫽ 31.7 ⫾ 1.2) and in
DC only (average Ct value ⫽ 31.4 ⫾ 0.5), and, as shown in
Fig. 4E, CX3CL1 expression was higher in the 3D model
with DC (average Ct value ⫽ 30.2 ⫾ 0.5) compared with the
3D model without DC. In addition, we analyzed the expression of IL-10, IL-13, IL-1␤, TNF, TGF-␤, and CXCL8.
Similarly to IL-4, IL-10 and IL-13 were undetectable in all
samples compared, and, although IL-1␤ (Ct values in the
range of 24.0 –27.9), TNF (Ct-values in the range of 26.9 –
29.8), TGF-␤ (Ct values in the range of 27.2–29.9), and
CXCL8 (Ct-values in the range of 19.6 –23.4) were abundantly expressed in all samples compared, no significant
differences were observed (data not shown).
Although, our results show that DC survive equally well in
the absence and presence of exogenous growth factors, this
raises the question of DC longevity in the tissue model. To
assess DC survival and to quantify DC numbers over time, we
utilized live confocal microscopy to image lung tissue models
that were set up with fluorescently labeled DC. Confocal
microscopy images of the developing 3D models were acquired at days 1, 3, 5, and 8 (Fig. 5, A and B). From the
microscopic projections, it is evident that DC in the tissue
model acquire a more elongated phenotype and that the layer
where DC are localized expands over time (Fig. 5A). Furthermore, DC numbers were enumerated in microscopic projections (Fig. 5B), and this revealed that the number of DC in
models was significantly lower at day 8 compared with day 1
(Fig. 5C). Thus this approach avoiding tissue digestion can be
used as a noninvasive method to observe and quantify the
longevity of DC in the 3D lung tissue model.
Next, we conducted experiments analyzing the expression of
HLA-DR, DC-SIGN, and CD11c, surface molecules typically
expressed by hematopoietic cells but not by nonimmune cells.
In the 3D model without DC, HLA-DR, DC-SIGN, and CD11c
mRNA were undetectable, whereas the mRNA expression was
abundantly expressed in DC only (average Ct value ⫽ 28.7 ⫾
1.4, 30.2 ⫾ 32.4 ⫾ 0.9, respectively) and in the 3D model with
DC (average Ct value ⫽ 29.5 ⫾ 1.7, 29.1 ⫾ 2.5, 33.7 ⫾ 1.1,
respectively). As shown in Fig. 5E, the expression of HLA-DR,
DC-SIGN, and CD11c was not significantly different comparing the expression between DC and the 3D model with DC,
thus confirming the presence of DC in the lung tissue model at
day 8 of culture and that DC surface molecule expression is not
significantly affected by the introduction into the lung tissue
model.
DC are widely distributed in the epithelial layer of the lung
tissue model. To further characterize and investigate the distribution of DC in the lung tissue model, we performed immunofluorescence staining of tissue sections using antibodies
against HLA-DR, DC-SIGN, and CD11c. This revealed that all
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Fig. 3. The lung tissue model produces proteins important for mucosal barrier integrity
and function. A and B: Immunofluorescence
analysis of lung tissue model for the production of E-cadherin (green) and claudin (red)
(A) and occludin (green) and claudin (red) (B)
is shown (original magnification ⫻200).
DAPI was used to reveal cell nuclei. C and
D: mucus-specific staining of the lung tissue
model is shown. Cryosections were stained
with Alcian blue after 5 (C) (original magnification ⫻200) and 10 (D) (original magnification ⫻630) days of air exposure (AE). All
results are representatives of at least 3 independent experiments. In each experiment, 6
tissue models were analyzed.
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three markers can be used in immunofluorescence analysis to
identify DC in the lung tissue model and confirmed that DC are
closely distributed to the epithelial layer within the lung tissue
model (Fig. 6, A–C). As shown in Fig. 6A, HLA-DR⫹ DC
could be detected within the epithelial layer. The immunofluorescence analysis also revealed that HLA-DR⫹DC-SIGN⫹
DC localized to the boundary between the epithelial and the
fibroblast/matrix layers (Fig. 6B). Interestingly, CD11c⫹DCSIGN⫹ DC were also identified on the apical side of the epithelial
layer (Fig. 6C). To confirm the expression CX3CR1, the receptor
for CX3CL1, on DC in the lung tissue model, tissue sections were
stained with antibodies against CX3CR1. As shown in Fig. 6D,
CX3CR1-positive cells could be detected in the lung tissue model.
This, together with previous reports establishing CX3CR1 and
CX3CL1 as important for the capacity of lung DC to interact with
the epithelial layer, may indicate that CX3CR1 and its ligand
contribute to the epithelial organization of DC observed in the
lung tissue model. Together, these data indicate that DC can easily
be identified in the tissue model and that DC are strategically
located underneath, within, and at the apical side of the epithelium
coherent with previous reports (24).
Although the data above indicate that DC are widely distributed in the epithelial layer of the model, it is possible that
the sectioning procedure introduces damage to the tissue and
possible artifacts regarding DC distribution. Therefore, we
established a modified model that could be imaged in real time
using multiphoton microscopy. To visualize the different interacting cell types, epithelial cells expressing tdTomato and CFSElabeled DC were utilized. Second, the model containing fibroblasts, fluorescent epithelial cells, and DC was monitored using
multiphoton microscopy analysis. As shown in Fig. 6E, live
imaging analysis of the lung tissue model confirmed that DC are
localized at the apical side of the epithelial cell layer of the lung
tissue model. Taken together, these data demonstrate that the
tissue model comprises a fibroblast collagen matrix rich in extracellular matrix proteins and a tightly joined stratified squamous
epithelium that sits on top of a continuous basement membrane as
well as DC that are closely associated to the epithelial layer.
The capacity of DC to produce chemokines is regulated by
the 3D organotypic model. Having established a lung tissue
model with human DC, functional assays were performed to
confirm that the 3D organotypic lung model contributed to
regulate chemokine production related to physiological conditions. We first determined whether the lung tissue model could
affect the capacity of DC to produce CCL18, a chemokine that
is constitutively expressed in lung at steady state and is
elevated in several human disorders, including various malignancies and inflammatory lung, skin, and joint diseases (20,
32). As shown in Fig. 7, expression of CCL18 was detectable
in the lung tissue model with DC, whereas CCL18 expression
was undetectable in the lung tissue model without DC. Furthermore, comparing CCL18 mRNA accumulation in the DC-
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Fig. 4. The lung tissue model supports survival of DC without the addition of exogenous growth factors, cerebrospinal fluid (CSF-2) and IL-4. To evaluate the
capacity of the lung tissue model to support survival of DC, immunohistochemistry analyses detecting human leukocyte antigen (HLA)-DR molecules were
performed on 8-␮m cryosections from lung tissue models cultured either in the absence or presence of CSF-2 and IL-4. (A–C) Immunohistochemical staining of
lung tissue model cryosections for HLA-DR is shown. Tissue sections from models containing DC and cultured in the absence (left) (A) or presence (middle) (B) of
exogenous growth factors or tissue sections from models without DC (C), were stained with an anti-HLA-DR antibody (brown) (original magnification ⫻200).
D: quantification of HLA-DR positive area within the lung tissue model is shown. To quantify the HLA-DR staining in tissue sections, acquired computerized image
analyses (ACIA) were used. Bar graphs indicate the amount of HLA-DR-positive staining in tissue sections from models absent of DC or models with DC that were
cultured in the absence or presence of exogenous growth factors. To determine whether cytokines and chemokines important for DC survival and function are expressed
in the lung tissue model, RNA was extracted and supernatants were collected from 3D models absent of DC, 3D models with DC, or control DC (cultured in medium
only). Analyses of mRNA levels were determined in tissue and cells using real-time RT-PCR. E: bar graphs indicate the relative expression of CSF-2, IL-4, and CX3CL1
mRNA accumulated in control DC or 3D models with or without DC. All results are representatives of at least 3 independent experiments. In each experiment, 6 tissue
models were analyzed. Data presented in bar charts are shown as mean values in triplicate from 1 representative experiment (⫾ SD). The statistical significance (1-way
ANOVA) of the indicated P values was determined: *P ⬍ 0.05, **P ⬍ 0.01, ***P ⬍ 0.005, ns ⫽ nonsignificant.
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containing 3D model to DC cultured in medium only revealed
enhanced CCL18 expression within the 3D model (Fig. 7A).
That CCL18 production by DC is regulated in the 3D model
was further supported by the fact that CCL18 protein was
readily detected in the culture supernatant of the lung tissue
model with DC (Fig. 7B). Next, we analyzed the capacity of
DC in the 3D model to produce CCL17 and CCL22, chemokines that are expressed constitutively in thymus but are barely
detectable in other peripheral tissues at steady state and that are
induced during inflammatory reactions and associated to human skin and pulmonary inflammatory diseases (13, 21). Compared with DC only, the expression of CCL17 and CCL22 was
relatively low in lung tissue models regardless of whether the
model was set up with or without DC (Fig. 7, C and E).
Furthermore, the levels of CCL17 and CCL22 protein were
reduced in the culture supernatants of 3D models with DC
compared with DC cultured in medium only (Fig. 7, D and F).
Together these data suggest that the 3D model has the capacity
to differentially regulate the production of chemokines in DC,
and this involves enhanced expression of CCL18 and a reduced
expression of CCL17 and CCL22.
Soluble components secreted from the lung tissue model are
responsible for the induction of CCL18 in DC. Next, we
focused our studies to determine whether soluble tissue-de-
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Fig. 5. Quantification of DC longevity in the
lung tissue model. DC labeled with the far-red
cell tracker, dimethyl dodecylamine oxidesyccinimidyl, were implanted in models and
visualized by live imaging confocal microscopy over time. A: representative 3D images
acquired at days 1, 3, 5, and 8 of culture are
shown. B: microscopy projections of 3D models that were used to enumerate DC within a
defined field are shown. Each defined area had
to include a minimum of 250 cells. C: bar
graphs indicate the total numbers of DC in the
lung tissue models. DC were quantified by
enumerating far-red fluorescently labeled propidium iodide-negative cells in each defined
area and multiplied with factor reflecting the
total 3D tissue model area. To determine expression of DC surface molecules, HLA-DR,
DC-specific ICAM-grabbing nonintegrin (DCSIGN), and CD11c, RNA was extracted from
3D models absent of DC, 3D models with DC,
or control DC (cultured in medium only). Analyses of mRNA were determined in tissue using
real-time RT-PCR. D: bar graphs indicate the
relative expression of HLA-DR, DC-SIGN, and
CD11c mRNA accumulated in control DC and
3D models with or without DC. All results are
representatives of at least 3 independent experiments. Data presented in bar charts are
shown as mean values in triplicate from 1
representative experiment (⫾ SD). The statistical significance (1-way ANOVA) of the indicated P values was determined: *P ⬍ 0.05,
ns ⫽ nonsignificant.
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rived components were responsible for the selective induction
of enhanced CCL18 production in DC. When the impact of
soluble tissue model-derived components on CCL18 production by DC was monitored, we found that exposure of DC to
medium conditioned with the lung tissue model induced enhanced CCL18 mRNA expression (Fig. 8A) and CCL18 protein secretion by DC (Fig. 8B). Thus the lung tissue model
secretes compounds involved in regulating CCL18 production by DC. To determine whether the composition of cells,
i.e., lung epithelial cells and fibroblasts, into an AE 3D
structure is necessary for the induction of enhanced production of CCL18 in DC, DC were also exposed to medium
conditioned with air-exposed epithelial cells or fibroblast as
well as epithelial cells and fibroblasts cultured as a mixed
(1:1) submerged monolayer. This revealed that only the
complete lung tissue model has the capacity to condition
medium that can be used to induce enhanced CCL18 expression in DC (Fig. 8C).
DISCUSSION
Tissue-specific niches, such as gut and lung mucosa, are
increasingly recognized as influencing a broad range of events
involved in immune homeostasis and inflammation as well as
the host response to infection (18, 30, 33). Although, in vitro
3D organotypic lung models have been established in the past
and are commercially available ranging from models of normal
airway epithelium to models of lung cancer, models with
relevant features and functions of the human airway wall
combined with human immune cells are lacking. Here, we have
established a 3D organotypic lung tissue model allowing studies of DC regulation by lung tissue specific cells. We first showed
that molecules involved in the formation of essential structures for
lung tissue function are produced and deposited in the model. We
then showed that the lung tissue model supports the survival of
DC independent of exogenous growth factors and that DC can
easily be identified in the tissue model being strategically located
underneath, within, and on top of the epithelium. We also showed
that the lung tissue model regulates production of the chemokines
CCL17, CCL18, and CCL22 in DC. Thus this lung tissue model
is well suited for a wide range of studies, including those on tissue
organization and remodeling as well as the role of tissue-derived
components regulating DC functional properties within the local
tissue microenvironment during homeostasis and in response to
pathogens.
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Fig. 6. DC are widely distributed throughout
the epithelial layer within the lung tissue
model. The distribution of DC in the lung
tissue model was visualized by immunofluorescence microscopy. A: tissue model sections were stained with an anti-HLA-DR antibody (green) (original magnification ⫻200).
B: tissue model sections were stained with an
anti-HLA-DR antibody (green) in combination with an anti-DC-SIGN antibody (red)
(original magnification ⫻200). C: tissue
model sections were stained with an antiCD11c antibody (green) in combination with
an anti-DC-SIGN antibody (red) (original
magnification ⫻200). Higher magnification
shows a DC in close association with epithelial cells at the mucosal surface (inset: original magnification ⫻1,000). D: tissue model
sections were stained with an anti-CX3CR1
antibody (green). The distribution of DC
within the lung tissue model was also visualized in live tissue using multiphoton microscopy. DAPI was used to reveal cell nuclei.
E and F: imaging was performed with models
composed of tdTomato expressing epithelial
cells (red) and carboxyfluorescein diacetate
succinimidyl ester-labeled DC (green). A top
view (E) and a side view (F) of the lung tissue
model are shown (original magnification
⫻200). All results are representatives of at
least 3 independent experiments.
DC REGULATION BY LUNG TISSUE-SPECIFIC CELLS
of diverse populations of immune cells from blood to tissue at
steady state and during inflammation and infection. The second
major outcome from this study is that this organotypic model
regulates the capacity of DC to produce one homeostatic
chemokine, CCL18, and two inducible chemokines, CCL17
and CCL22. CCL18 is a constitutively expressed chemoattractant produced by immune cells and predominantly associated
to lymphnode and lung tissue, and that is detected at high
levels in human serum at homeostatic conditions (20). The
influence of tissue in the involvement of regulating production
of CCL18 in DC was confirmed by demonstrating that DC in
the organotypic model make more CCL18 than those cultured
under conventional conditions. This finding, therefore, supports the fact that the lung tissue model microenvironment
induces changes to DC and selectively modulates the production of chemokines resembling physiological conditions (13,
20, 21). Further support of a functional distinction between DC
in the 3D organotypic model compared with DC cultured under
conventional conditions comes from our findings that implanting DC in the model reduced the levels of CCL17 and CCL22
detected. CCL17 and CCL22 are not detectable at steady state
but are induced during inflammatory reactions and associated
to human skin and pulmonary inflammatory diseases (16, 41),
thus implicating that specific tissue is able to indirectly exert
immune regulatory function via their influence on DC function
locally. Furthermore, the results from our study also demonstrate that soluble components secreted by 3D organotypic
models are responsible for the enhanced production of CCL18
in DC. Although, differential expression of cytokines and
chemokines by DC in response to monolayer of lung epithelial
cells has been observed (26, 29), the effect of 3D tissue models
on DC function is limited. Therefore, we investigated whether
there are differences between the 3D organotypic model, AE
epithelial cells, AE fibroblasts, or submerged monolayer culture of epithelial cells and fibroblasts in terms of altering the
capacity of DC to produce CCL18. Medium conditioned with
the 3D organotypic model was more potent than conditioned
medium from the single cell cultures or the monolayer culture,
Fig. 7. Chemokine expression by DC is regulated by the lung tissue microenvironment. To determine whether chemokine production is modulated in DC in
response to the lung tissue microenvironment, RNA was extracted and supernatants were collected from 3D models absent of DC, 3D models with DC, or
control DC (cultured in medium only). Analyses of mRNA and protein levels
were determined in tissue and supernatants using real-time RT-PCR and
ELISA, respectively. A and B: bar graphs indicate the relative expression of
CCL18 mRNA accumulated in control DC or 3D model (A) and the concentration of CCL18 in control DC or 3D model culture supernatants. C–F: bar
graphs indicate the relative expression of CCL17 (C) and CCL22 (E) mRNA
accumulated in control DC or 3D model culture supernatants and the concentration of CCL17 (D) and CCL22 (F) in control DC and 3D model culture
supernatants. The lung model was set up in the absence of CSF-2 and IL-4, and
culture supernatants were conditioned for 48 h before protein quantification.
Control DC were cultured in the absence of CSF-2 and IL-4 for 24 or 48 h
before RNA and protein analyses. Before RNA extraction, control DC, 1.5 ⫻
105 (which is a similar number of cells that was implanted in each 3D model
during set up), were mixed with 3D models composed of fibroblasts and
epithelial cells only. All results are representatives of 3 independent experiments, and DC from a total of 6 donors have been used. Data presented in bar
charts are shown as mean values of mRNA or protein levels in triplicate from
the 3 independent experiments (⫾ SD). The statistical significance (1-way
ANOVA) of the indicated P values was determined: *P ⬍ 0.05, **P ⬍ 0.01,
***P ⬍ 0.005.
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The finding that our human 3D organotypic lung model has
the capacity to support survival and allow functional studies of
human DC has important implications for increasing our understanding of local immune regulation in lung tissue. This novel 3D
organotypic lung model composed of human lung epithelial cells
and fibroblasts in combination with human DC shares similarities
with real tissue, including the overall structure and deposition of
matrix proteins essential for lung tissue function and forms a
functional mucosal barrier including production of proteins necessary for tight junction and adhesion junction formation. In this
context, the 3D AE tissue model provides a unique system in
which to further elucidate mechanisms of tissue organization and
remodeling at steady state and in response to external stimuli,
including viral and bacterial infection.
Coordinated expression of chemokines by nonimmune and
immune cells is essential in mediating adhesion and migration
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in promoting enhanced CCL18 production, suggesting that the
3D organotypic model secrets additional factors contributing to
this process, thus indicating that the complex structure of the
lung tissue model favors the production of factors involved in
regulating CCL18 production in DC under homeostatic conditions. This finding is quite intriguing and merits further studies.
In addition to 3D organotypic tissue models, various alternative cell culture systems are available as tools for studying
the impact of local tissue on immune cell function. Each
system has its distinct advantages and disadvantages, and the
choice of system depends on various factors, including the
experimental question being addressed and the cost, training,
and expertise required to successfully establish, validate, and
apply the model. One common technique involves utilizing
monolayers that are permitting infection but are usually based
on a single nonpolarized cell type in the absence of 3D cellular
interactions. Culture in Transwells allows culturing of monolayers on a permeable membrane that, unlike traditional monolayers systems, allows the cells to develop an apical-basal
polarity. These polarized surfaces can be independently exposed to different factors, including pathogens and toxins, and
thereby exposing the cells in a physiologically relevant setting
and taking into account transepithelial migration (10). One
disadvantage of the method is that these cultures are often
based on one type of cell only. Also, such models lack
extensive 3D cellular interactions that are achieved with 3D
matrix scaffold cultures, which are crucial for normal tissue
function as well as infection processes (8).
The engineering of organotypic models involves implanting
of cells or tissue into a 3D matrix scaffold composed of
collagen, extracts of extracellular matrix, synthetic or semisyn-
thetic materials, or a combination of these materials. However,
models with relevant features and functions of the human
airway wall combined with human immune cells are lacking.
In this study, therefore, we sought to develop a novel human
3D lung tissue model that utilizes appropriate human cell
populations, including bronchial epithelial cells, fibroblasts,
and human immune cells. In our model epithelial (16HBE) and
fibroblast (MRC-5) cell lines are used, as they allow for
reproducibility compared with using potentially impure primary cells. The choice of this epithelial line was based on the
fact that 16HBE cells resemble primary lung epithelial cells
(38) and thus are a valuable and reproducible model of normal
lung epithelial tissue (7). Although, quite laborsome, this
relatively inexpensive approach provides a platform that also
allows studies in conjunction with manipulation of gene expression in tissue-specific nonimmune cells. Thus the model is well
suited for studies on tissue-specific regulation of human DC
functional properties and migratory behavior in a physiologically
relevant setting previously not achievable. Furthermore, the model
allows exposure of cells under physiological conditions where
important aspects of host-pathogen interactions can be monitored
in real time. In conclusion, we have established a human 3D
organotypic lung model in which tissue-specific mechanisms that
regulate DC function locally during steady state and in response to
inflammation can be studied. By further understanding the mechanisms whereby specific tissue niches control the functional properties of DC, we might uncover potential targets for manipulating
the DC function in therapeutic settings to restore tissue homeostasis not only in acute and chronic infectious diseases but also
other chronic inflammatory diseases.
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Fig. 8. Chemokine expression by DC is regulated by soluble components secreted from
the 3D organotypic model. To determine
whether chemokine production is modulated
in DC in response to soluble compounds produced by the tissue-specific cells in the 3D
organotypic model, supernatants were collected from tissue models absent of DC, AE
epithelial cells, AE fibroblasts, or submerged
monolayer cultures of epithelial cells and fibroblasts. DC were stimulated with culture
supernatants for 24 – 48 h followed by the
detection of mRNA accumulation and protein
secretion real-time RT-PCR and ELISA, respectively. A: bar graphs indicate the accumulation of CCL18 mRNA in DC stimulated
with culture supernatants from the 3D organotypic model or medium only. B: bar graphs
indicate the amount of CCL18 protein accumulated from DC stimulated with culture supernatants from the 3D organotypic model or medium only. C: bar graphs indicate the accumulation of CCL18 mRNA in DC stimulated for
24 h with medium only or culture supernatants
(48 h-conditioning) as indicated. Before stimulation DC were cultured in the absence of GMCSF and IL-4 for 24 h. All results are representatives of at least 3 independent experiments,
and DC from a total of 5 donors have been used.
Data presented in bar graphs are shown as mean
values of mRNA levels in triplicate from the 3
independent experiments (⫾ SD). The statistical
significance (1-way ANOVA) of the indicated P
values was determined: *P ⬍ 0.05, **P ⬍ 0.01.
DC REGULATION BY LUNG TISSUE-SPECIFIC CELLS
ACKNOWLEDGMENTS
We thank Dr. Oscar Hammarfjord for critical review of the manuscript.
GRANTS
This work is supported by grants from the Swedish Research Council, the
Karolinska Institutet, Stockholm County Council, The Swedish Fund for
Research Without Animal Experiments, Erik och Edit Fernströms Foundation
(to M. Svennson). M. Coles is supported by funding from the Medical
Research Council (G0601156). A. T. Nguyen Hoang is a recipient of a
Karolinska postgraduate studentship. J. Juarez and L. Bosnjak are recipients of
Marie Curie International Incoming Fellowships.
DISCLOSURES
No conflicts of interest, financial or otherwise are declared by the authors.
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