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RESEARCH Open Access
Surfactant Protein D modulates allergen particle
uptake and inflammatory response in a human
epithelial airway model
Carsten Schleh
1,2*
, Barbara M Rothen-Rutishauser
3,4
, Fabian Blank
3
, Hans D Lauenstein
1,2
, Matthias Nassimi
1,5
,
Norbert Krug
1
, Armin Braun
1
, Veit J Erpenbeck
1,2
, Peter Gehr
3
and Jens M Hohlfeld
1,2
Abstract
Background: Allergen-containing subpollen particles (SPP) are released from whole plant pollen upon contact
with water or even high humidity. Because of their size SPP can preferentially reach the lower airways where they
come into contact with surfactant protein (SP)-D. The aim of the present study was to investigate the influence of
SP-D in a complex three-dimensional human epithelial airway model, which simulates the most important barrier
functions of the epithelial airway. The uptake of SPP as well as the secretion of pro-inflammatory cytokines was
investigated.
Methods: SPP were isolated from timothy grass and subsequently fluorescently labeled. A human epithelial airway
model was built by using human Type II-pneumocyte like cells (A549 cells), human monocyte derived
macrophages as well as human monocyte derived dendritic cells. The epithelial cell model was incubated with SPP
in the presence and absence of surfactant protein D. Particle uptake was evaluated by confocal microscopy and
advanced computer-controlled analysis. Finally, human primary CD4
+
T-Cells were added to the epithelial airway
model and soluble mediators were measured by enzyme linked immunosorbent assay or bead array.
Results: SPP were taken up by epithelial cells, macr ophages, and dendritic cells. This uptake coincided with
secretion of pro-inf lammatory cytokines and chemokines. SP-D modulated the uptake of SPP in a cell type specific
way (e.g. increased number of macrophages and epithelial cells, which participated in allergen particle uptake) and
led to a decreased secretion of pro-inflammatory cyt okines.
Conclusion: These results display a possible mechanism of how SP-D can modulate the inflammatory response to
inhaled allergen.
Keywords: Allergen Particle, Subpollen Particles, SPP, Surfactant Protein D, SP-D, Cytokines
Background
Over the past 30 years asthma prevalence has markedly
increased. A high percentage of asthmatics is sensitized
against pollen [1]. Whole pollen are large in size (up to
50 μm) so that they preferentially deposit in the upper
airways with a very low fra ction reaching the deeper
lung upon inhalation. However, allergen l oaded subpol-
len particles (SPP, also named pollen starch granules
(PSG)) are released from whole pollen upon contact
with water or e ven at high humidity and their release is
associated with an increased outcome of asthma symp-
toms e.g. during thunderstorms [2]. Because of their size
(d < 5 μm) SPP can deposit in the al veolar region of the
lung subsequent to inhalation [3]. Since this region is
completely covered with the pulmonary surfactant layer
which is defined as a surface-active complex of lipids
and proteins, deposited particles are directly translo-
cated to the aqueous hypophase due to wetting forces
upon impingement [4, 5]. Importantly, the hypophase is
the compartment of hydrophilic surfactant protein D
(SP-D) and it is known that SP-D is able to bind to SPP
[6]. SP-D belongs to the family of the collectins
* Correspondence: carsten.schleh@web.de
1
Fraunhofer Institute of Toxicology and Experimental Medicine, Nikolai-Fuch-
Str. 1, 30635 Hannover, Germany
Full list of author information is available at the end of the article
Schleh et al. Respiratory Research 2012, 13:8
http://respiratory-research.com/content/13/1/8
© 2012 Schleh et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
(collagen containing lectins) and is built of 12 mono-
mers (43 kDa) each consisting of a N-terminal region, a
collagen-like domain, a neck re gion and a globular head
carbohydrate recognitio n domain (CRD) [7]. Three of
these monomers cluster to trimers (~ 130 kDa) and four
of these trimers assemble to SP-D, which is a dodeca-
mer (~ 520 kDa) with a crucifix form. In addition, SP-D
can oligomerize into larger m ultimers (> 1 MDa) [8].
Importantly, binding of SP-D to SPP can lead to an
increased phagocytosis of the SPP by alveolar macro-
phages (MO) [6]. In addition, SP-D modulate s the inter-
action of SPP and airway epithelial cells (EC) as shown
in a previous study [9]. Besides MO and EC, dendritic
cells (DCs) which realize as sentinels and most compe-
tent antigen-presenting cell a surveillance network in
the pulmonary tissues are among the key players in
initiating and maintaining allergic diseases [10]. How-
ever, little is known about their uptake potential of SPP
and the influence of S P-D on the down-stream immu-
nological reactions. A previous study described, that SP-
D reduced the number of SPP-positive dendritic cells
(DCs) [11].
Most of the studies, including the above mentioned,
which focused on the interaction of SP-D and allergen
particles or other pathogens were accomplished in in
vitro-monocultures which lack important cell-to-cell
interactions to resemble the in vivo situation. In vivo,
cells are able to communicate directly and indirectly
with each other in order to orchestrate a specific or
unspecific immuneresponse[12-15].Toaccountfor
this, we studied the uptake of SPP from timo thy grass
( Phleum pratense)aswellastheinfluenceofSP-Din
cells within an epithelial airway model, consisting of
human monocyte-derived macrophages (MDM), ECs,
and human monocyte-derived dendritic cells (MDDC)
[16-18]. This complex cell culture model warranted
maximal interaction between the participating cells as it
has been shown upon exposure of the three cell types to
1 μ m polystyren e particles [19]. By using laser scanning
microscopy (LSM) and advanced computer-controlled
analysis, t he percentage of cells which participated in
SPP-uptake, as well as the absolute number of allergen
particles within single cells was investigated. To evaluate
the patho-physiological consequence of the SPP-uptake
and the modulation by SP-D, autologues CD4
+
-T-cells
were isolated from blood of allergic donors, added to
the epithelial airway mod el, and inflammatory mediators
were measured after further incubation.
Methods
Material
Rat recombinant SP-D (SP-D) was purified by maltose
affinity chromatography from the media supernatant of
cultured Chinese hamster ovary cells stably transfected
with a full-length rat SP-D cDNA clone as described
previousl y [20]. Timothy grass (Phleum pratense) pollen
were obtained from Allergon (Ängelholm, Sweden). All
other reagents, unless otherwise specified, were pur-
chased from Sigma Chemical (Deisenhofen, Germany).
All subjects who donated blood gave their w ritten
consent after being fully informed about the purpose
and nature of the studies which were approved by the
Ethics Committee of Hannover Medical School.
Subpollen particles
SPP were isolated from timothy grass pollen as
described previously [6]. 300 mg of pollen were shaken
and vortexed in 40 ml of deionized, a utoclaved water
for 3 min. Whole pollen and pollen fragments were
then separated by centrifugation at 50 g for 4 min. The
supernatant was filtered (5 μmfilter,VWRInterna-
tional, Hannover, Germany) and centrifuged twice at
2500 g for 10 min. The resulting pellet was resuspended
in 1 ml of sterile NaHCO
3
(0.1 M) for fluorescence
labelling or phosphate buffered saline (PBS) for direct
use in the experiments. To determine the number of
SPP, an aliquot was diluted i n PBS (1:100) and then
counted in an improved Neubauer chamber.
Immediately after the isolation procedure, SPP were
fluorescently labelled with Alexa Fluor 4 88 fluorescent
dye (Molecular Probes, Eugene, OR). For the staining
procedure an amount of 1 × 10
9
-2×10
9
SPP in 1 ml
NaHCO
3
(0.1 M) was used. The suspension was trans-
ferred into the vial of reactive dye and rotated for 1 h at
room temper ature in the dark. Sterile PBS (14 ml) was
added, centrifuged at 2500 g for 12 min and the pellet
was resuspended in 1 ml of PBS. The SPP were counted
under fluorescence light in an improved Neubauer
chamber.
During this study, measurement of Lipopolysaccharide
(LPS) contamination of the SPP was not performed.
A549 culture
A549 cells were obtained from the American T ype Cul-
ture Collection (ATCC) and cultured in RPMI 1640
Medium (Cambrex Bio Sciences, Walkerswille, MD)
supplemented with 10% heat-inactivated foetal calf
serum (FCS) and 1% p enicillin-streptomycin in a 37°C
humidified atmosphere with 5% CO
2
.
Triple cell co-cultures
Cultures were prepared as previously described [16] and
as shown in Figure 1A. Briefly, A549 cells (passage 10-
40) were grown on cell culture inserts (surface area of
4.2 cm
2
, pores of 3.0 μm in diameter, high pore density
PET membranes for 6-well plates; BD Biosciences, Basel,
Switzerland). Macrophages and dendritic cells were
derived from human blood monocytes as described
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before [16]. Briefly, peripheral blood monocytes were
isolated from buffy coats (blood donation service, Bern,
Switzerland) or whole blood (Fraunhofer ITEM, Hann-
over, Germany) and cultured in the same medium as
used for the epithelial cells except for the supplemen-
tation of 5% human serum (blood donation service
Bern, Switzerland and Invitrogen, Karlsruhe, Germany)
instead of 10% foetal calf serum. For the generation of
MDDC the monocytes were cultured for 7d in medium
supplemented with 34 ng/mL IL-4 (Sigma, Fluka-
Chemie GmbH, Buchs, Switzerland) and 50 ng/mL
GM-CSF (R&D Systems, Oxon, UK), whereas MDM
were obtained from peripheral blood monocytes cul-
tures w ithout additional supplements for 7 d ays. A549
epithelial cells were cultured for 7 days before MDM
were added on top of the epithelial monolayer and
finally MDDC were added underneath the insert mem-
brane. The triple cell co-cultures were kept overnight
in medium supplemented with 1% L-Glutamine, 1%
penicillin/streptomycin, and 5% heat inactivated
(pooled)humanserumat37°Cin5%CO
2
humidified
atmosphere. Triple cell co-cultures were incubated for
8hourswith10×10
6
Alexa488 labelled SPP which
were added to the apical chamber for uptake quantifi-
cation or with unlabelled SPP for subsequent cytokine/
chemokine determination.
Quadruple cell co-cultures
The isolation of immune cells was done from blood of
Phleum pratense sensitized atopic humans for in vitro
experimentation. Subjects had to have a history of
allergy to g rass pollen and a positive skin prick test for
Phleum pratense pollen at or within 12 months prior to
their visit. Autologous CD4
+
-T-cells were isolated
according to the manufactures instructions (CD4
+
-T-cell
Isolation Kit II, Miltenyi Biotec, Bergisch Gladbach, Ger-
many) and subsequently stored for 1 week at -80°C.
After 8 hours incubation of the triple cell co-cultures
with unlabelled S PP and SP-D, thorough washing steps
with phosphate buffered saline solution (PBS) were per-
formed. The membrane with the triple cell co-cultures
was excised, turned around, and placed in a 12-well
plate so that the MDDC resided o n the upper side of
the membrane (Figure 1B). After thawing the autologous
T-cells were added to the epithelial airway model (DC-
T-cell proportion: 1:10) in 2 ml medium and incubated
for 72 hours.
Cell labelling and fixation
Cells were wa shed in PBS and fixed for 15 min a t room
temperature in 3% paraformaldehyde in PBS. Fixed cells
were treated with 0.1 M glycine in PBS for 5 minutes
and permeabilized in 0.2% Triton X-100 in PBS for 1 5
min. The cells were incubated with primary and second-
ary antibodies for 60 min each at room temperature.
Preparations were mounted in PBS:glycerol (2:1) con-
taining 170 mg/mL Mowiol 4-88 (Calbiochem, VWR
International AG).
Antibodies were diluted in PBS as follows: mouse anti-
human CD14 1:20 (Clone UCHM-1, C 7673, Sigma,
Deisenhofen, Germany), mouse anti-human CD86 1:20
(Clone HB15e, 36931A, PharMingen, BD Biosciences),
goat anti-mouse cyanine 5 1:50 (AP124S, Chemicon,
VWR International AG, Life Sciences, Lucerne,
Figure 1 Epithelial airway model. A) Creation of the triple cell co-culture model. B) Membrane with the epithelial airway model was excised,
turned around and CD4+-T-cells were added.
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Switzerland), and phalloidin-rhodamine 1:100 (R-415,
Molecular Probes, Invitrogen AG, Basel, Switzerland).
Laser scanning microscopy and image restoration
A Z eiss LSM 510 Meta with an inverted Zeiss micro-
scope (Axiovert 200 M, Lasers: H eNe 633 nm, HeNe
543 nm, and Ar 488 nm) was used. Image processing
and visualization was done using IMARIS, a 3D multi-
channel image processing software for confocal micro-
scopic images (Bitplane AG, Zurich, Switzerland).
Particle quantification
After image acquisition the total particle number per
scan was counted with the particle tracking software
Diacount
®
(Semasopht, Lausanne, Switzerland) as
already described for polystyrene particles o f d ifferent
sizes [21,22] and iron-oxide hybrid nanoparticles [23].
For each experi mental sample, cells were randomly
scanned with the LSM. The particles were counted
within individually defined cell types, which were
labelled with specific cell surface markers (CD14 for
MDM, CD86 fo r MDD C, and F-Actin for the EC). One
observer performed the particle quantification.
Cytokine/Chemokine determination
Supernatants were taken after 8 hours incubation with
unlabelled SPP and further 72 hour incubation with
CD4
+
-T-cells in fresh medium. All cytokines and che-
mokines were measured by bead-based protein quantifi-
cation. A Bioplex 200 System (Biorad, München,
Germany) and a Milliplex bead kit (Millipore, Schwal-
bach, Germany) were used according to the manufac-
turer’s instructio ns. Since the values of Inter leukin (IL)-
8 w ere above the limit of quantifications, cell culture
supernatants were diluted and IL-8 was measured by
enzyme linked immunosorbent assay (Duoset, R&D
Bioscience, Wie sbaden-Nordenstadt, Germany) ac cord-
ing to the manufacturer’ s instructions. The limit of
detection was the lowest standard value of the respective
cytokine/chem okine. The limit of quantification was the
lowest standard value where the duplicates had a coeffi -
cient of variation < 0.2 and a mean which was at least 5
times higher than the blank value.
Uptake Index
To assess the effect of SP-D, the percentage of positive
cells was determined and the number of SPP per cell
was counted. In addition, we calculated an uptake index
(UI), which considered the percentage of cells contain-
ing particles multiplied by the number of SPP inside sin-
gle cells. The UI was estimated from 6 individual
experiments as described by the following formula:
UI =
C
(
p
)
∗ P
(
c
)
C
(p)
: Percentage of cells c ontaining particles. P
(c)
:
Number of particles inside cells.
Statistical analysis
Values are given as means ± SEM. Statistical analysis
was performed using GraphP ad Prism
®
, Version 4.03.
Statistical comparison of the means was performed by
ANOVA, followed by a Bonferroni correction. P-values
< 0.05 were considered to be significant.
Results
Uptake of subpollen particles
SPP were found intr acellulary in MDM (Figure 2A), EC
(Figure 2B), and MDDC (Figure 2C), within the human
epithelial airway model. Importantly, SP-D was able to
modulate this uptake as it can be seen exemplarily in
Figure 3.
A detailed quantification revealed t hat 43.0 ± 9.8% of
the MDM took up 21.5 ± 7.2 S PP per cell after 8 ho urs
incubation with 10 million SPP. Furthermore, 3.3 ±
0.7% of the EC took up 2.9 ± 0.3 SPP, an d 47.9 ± 13.0%
of the MDDC internalized 31.2 ± 18.2 SPP on average
(Figure 4). A significantly increased percentage of parti-
cle-positive cells was found after co-incubation of the
SPP with SP-D (Figure 4A). Whereas 1 μg SP-D/ml did
not lead to a significant modulation, 10 μgSP-D/ml
incre ased the percentag e of SPP- positive MDDC to 75.4
± 4.1% (p < 0.05) and the percentage of SPP-p ositive EC
to 18.7 ± 4.1% (p < 0.01). The percentage of MDDC,
which were positive for SPP, was not significantly
modulated by SP-D.
In contrast, the number of SPP, which were taken up
by individual MDM and MDDC, was lower after co-
incubation with SP-D compared to control conditions
without SP-D (Figure 4B). SP-D (1 and 10 μg/ml)
reduced t he number of intracellular SPP to 15.5 ± 5.2
and 10.7 ± 3.8 SPP, respectively. The number of SPP in
MDDC was decreased to 19.9 ± 11.7 after co-incubation
with 1 μg/ml S P-D a nd to 10.1 ± 2.8 SPP after incuba-
tion with 10 μg/ml SP-D. The amount of SPP in EC
stayed low and unchanged after co-incubation with SP-
D [1-10 μg/ml].
The uptake index (UI) for SPP in MO after incubation
with 10 million SPP, i.e. 685.4 ± 193.2, stayed nearly
constant after co-incubation with increasing concentra-
tions of SP-D (Table 1). No dose-response was detected.
Interestingly, the UI of EC was higher after co-incuba-
tion with 1 μg/ml and 10 μg/ml SP-D and increased
from 9.9 ± 2.5 to 55.8 ± 32.8 and 64.1 ± 26.4, respec-
tively. However, these increases were not significant. In
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contra st, the UI of MDDC decreased from 1982 ± 1346
to 1018 ± 455.9 and 570.6 ± 232.2 after co-incubation
with SP-D [1 and 10 μg/ml]. Again, these changes were
not significant.
(Pro-) Inflammatory Response
Incubation with 10 million SPP for 8 hours increased
secretion of IL-8 when cells were incubated for addi-
tional 72 hours with fresh medium in the presence of
CD4
+
-T-cells. The baseline value of 61.3 ± 11.3 ng/ml,
measured in the supernatants of the cells alone, was
increased to 103.3 ± 24.3 ng/ml. Importantly, co-incuba-
tion wit h SP-D during the 8 hour-particle exposure per-
iod decreased IL-8 secretion significantly (SP-D 1 μg/ml:
56.0 ± 9.8 ng/ml and SP-D 10 μg/ml: 50.5 ± 10.5 ng/ml/
Figure 5).
In addition, cells of the epithelial airway model
secreted various other cytokines and chemokines (Table
2). After the 72 hours incubation period with fresh
medium which followed the 8 hour i ncubation perio d
with 10 million SPP increased mediator-levels were
recorded. SPP exposure significantly increased secretion
of IL-1 alpha compared to untreated cells (108.2 ± 22.0
pg/ml versus 170.6 ± 40.6 pg/ml) and Macrophage
Inflammatory Protein 1 (MIP-1) beta (1211 ± 105.6 pg/
ml versus 1408 ± 143.6 pg/ml). An increase by trend
was observed for Granulocyte-Colony Stimulating Factor
(G-CSF), Tumor Necrosis Factor (TNF)-alpha, and IL-6.
In contra st to IL-8, the secretion of these mediators was
not significantly modulated by co-incubation with sur-
factant protein D [1-10 μg/ml].
Discussion
The present study describes for the first time t he influ-
ence of SP-D on ce llular u ptake of allergen particles
such as SPP within a complex lung cell culture model
and the secretion of cytokines/chemokines. Our data
show that SP-D increased the number of MDM and EC,
Figure 2 MDM(A), EC (B), as well as MDDC (C) are able to internalize subpollen particles (SPP). In each pictur e, three dimensions of the
cells along the white lines are shown. The intersections display uptaken SPP. The pictures were made by a confocal laser scanning microscope.
Green: SPP (Alexa 488); red: F-Actin (Rhodamine phalloidin); blue: CD14 (A), and CD86 (C). 1 represents xy sections, 2 yz and 3 xz.
Schleh et al. Respiratory Research 2012, 13:8
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which participated in allergen particle uptake. Interest-
ingly, the num ber of SPP per single cell did not increase
upon i ncubation with increasing SP-D concentration.
Using an uptake index (UI) that considered both, the
percentage of c ells containing particles as well a s the
number of SPP inside single cells, we demonstrated that
the total number of SPP stayed constant in MDM, was
increased in EC, and was decreased in MDDC.
Figure 3 Pictures displaying the strong modulation of surfactant protein D (SP-D) on distribution of subpollen particles (SPP) within a
human epithelial airway model. Left pictures show an overview of the upper side of the epithelial airway model after incubation with SPP
with or without SP-D. Right pictures show internalized and attached SPP after masking of cell borders. A) Epithelial airway model after 8 hours
incubation with 10 Million SPPB) Epithelial airway model after 8 hours incubation with 10 Million SPP+ 10 μg/ml SP-D. Green: SPP (Alexa 488);
red: F-Actin (Rhodamine phalloidin); blue: CD14. Arrows on the left pictures point to SPP which are attached on the surface of the EC, arrows on
the right pictures point to SPP inside cells.
Figure 4 Influence of sur factant protein D (SP-D) on (A) percentage of cells, within an epithelial airway model, which participated in
uptake of subpollen particles (SPP) and (B) on number of intracellular subpollen particles (SPP) in single cells. Analysis was performed
after 8 hours incubation of the epithelial airway model with 10 million SPP by confocal microscopy. CO: Control; MDM: Monocyte derived
macrophages; EC: Epithelial Cells; MDDC: Monocyte derived dendritic cells Means of at least 5 experiments ± SEM are shown. * p < 0.05; ** p <
0.01
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Incubation with SPP increased secretion of (pro-)inflam-
matory cytokines and chemokines. SP-D inhibited the
IL-8 release from the cells.
Little is known about the uptake of naturally occurring
allergen particles by resident lung cells. In addition,
although SP-D is one of the first proteins which comes
into contact with inhaled particles and thereby modulates
immune responses [24], only few data exist describing the
effects of a SP-D-particle interaction mainly leading to a
increased percentage of alveolar macrophages which took
up allergen particles [6,11]. However, the same study also
showed that SP-D reduced the number of SPP-positive tis-
sue macrophages and DCs 24 hours after in vivo instilla-
tion of the particles [11]. These results highlight the
potential differences between effects observed in vitro and
in vivo and further emphasize the need of complex in vitro
systems which may much better mimic specific in v ivo
situations. In fact, our results show a decreased UI for the
MDDC which would be in line with the results of the
study of Winkler and co-workers considering a substantial
amount of particle clearance during the longer observation
period they used [11]. Within the present study, it is
important to note, that the measurement of particle quan-
tification was only performed by a single observer.
Although the software Diacount
®
does hardly al low for a
subjective counting, t his should be taken into considera-
tion. Importantly, we were able to reliably determine SPP
uptake in contrast to binding and focused only on the
intracellular SPP. Recently we showed that SP-D increased
the amount of human primary bronchial epithelial cells
with attached SPP [9]. It is further known that the uptake
of particle-like surfactant-aggregates in type II pneumo-
cytes is increased upon co-incub ation with SP-D [25]. In
accordance, we found an increased amount of EC which
took up SPP and also an increased UI upon in cubation
with SP-D. Importantly, in a previous study [9], we found
that SP-D was able to modulate the interaction of SPP
with human primary bronchial epithelial cells but not with
A549 cells. Interestingly, the A549 cells, incorporated in
the present epithelial airway mode l, reacted much more
sensitive upon contact with SP-D and SPP compared to
the A549 in vitro monocultures [9]. Hence we may specu-
late that contact to other lung cells such as immune cells
modulates the A549 cells to react more similar than
human primary epithelial cells.
Incubation with 10 million SPP led to an increased (pro-
)inflammatory response within the epithelial airway model
after further 72 hours incubation with fresh medium com-
pared to cultures without SPP. This clearly indicates an
inflammatory effect of SPP that may also occur in the lung
after a single inhalation of allergen particles and that may
be independent of allergic sensitization to the particles.
The increased secretion of several cytokines (e.g. IL-8)
determines a pro-inflammatory action of the SPP which
was previously observed for various other allergens in dif-
ferent experimental systems [26-30]. Interestingly, some of
these mediators are even released by cells upon contact to
iner t particles. It is e.g. known, that polystyrene parti cles
induce the secretion of TNF-alpha [31] or IL-8 [32].
Furthermore, SPP are a natural material and are conse-
quently contaminated by LPS. This contamination is of
course at least partly responsible for the secretion of
inflammatory cytokines. However, it was the aim of the
study to investigate the allergen particles as they occur in
the nature and not only by means of a purified laboratory
situation. Hence we conclude that the secretion of (pro-)
inflammatory mediators belongs to all three parameters:
the nature of the allergen itself, the particulate body of the
allergen particles, and possible LPS contamination.
Importantly, an allogenic response within the co-c ul-
ture model can be excluded. With respect to IL-8, we
Table 1 Modulation of Uptake Index by surfactant
protein D (SP-D)
Uptake Index 0 μg/ml SP-D 1 μg/ml SP-D 10 μg/ml SP-D
MDM 685.4 ± 193.2 511.3 ± 212.7 815.5 ± 289.9
EC 9.9 ± 2.5 55.8 ± 32.8 64.1 ± 26.4
MDDC 1982 ± 1346 1018 ± 455.9 570.6 ± 232.2
Figure 5 Secretion of Interleukin (IL)-8 into supern atants of an
epithelial airway model after 8 hours incubation with 10
million subpollen particles (SPP) ± surfactant protein D (SP-D)
and further 72 h incubation with fresh medium without
particles and proteins. IL-8 was measured by enzyme linked
immunosorbent assay. Values are means ± SEM from of least 5
experiments. # p < 0.05; ## p < 0.01; Detection limit was 3.2 pg/ml;
Limit of quantification was 64 pg/ml.
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observed cytokine se cretion after measuring just the cell
culture system without any stimulus. However, after
incubating the cell-culture system with SPP, we found a
significant increase of IL-8. Hence we conclude that this
increase is not the result of an allogenic stimulation.
The addition of SP-D decreased this SPP-modulated
increase of IL-8 which further supports the notion of
being no allogenic effect.
SP-D led to a significantly decreased level of IL-8 in
the supernatant in the cell cultures after incubation.
This is in co ntrast to a former study with primary bron-
chial epithelial cells in monocultures. It has been
observed that epithelial mono-cultures and triple cell
co-cultures react differently upon exposed to various
particles e.g. diesel exhaust particles, titanium dioxide as
well as single-wall carbon nanotubes [33,34]. We
hypothesize that there is a synergistic effect due to the
interaction of the three cell types (EC, MDM and
MDDC) that reduce the adverse effects of the xenobio-
tica. It is important to note that different cell types,
included in a complex cell culture model, are better at
simula ting the real situation in the lung than mono-cul-
tures.Hencewebelieve,that the present results simu-
late more realistic the in vivo situation. In a former
study performed with fixed co-cultures of MDM placed
on top and MDDC placed below a monolayer of EC and
exposed to 1 μm polystyrene particles, we wer e able to
visualize frequent interactions between these two cell
types. MDM and MDDC were found to extend cytoplas-
mic processes across the epithelial barrier building cell-
cell contacts, and particles were found in MDM and
MDDC, some of them near cell-cell contacts [ 35].
Another study revealed the expression of tight junction
and adherens junction protein in MDM and MDDC
which was suggested to prese rve the epithelial integrity
in a trans-epit helial network maintained b y both
immune cells i n order to capture and translocate
inhaled particulate antigen through the epithelial lung
barrier [36]. These ways of communication have to be
investigated in more detail in further studies in order to
understand cellular interactions in cell culture models.
Importantly, the cell cultures within these experiments
were observed under a light microscope after the end of
each experiment. Thereby, we could clearly see the nor-
mal cell shape with no signs of apoptotic blebbing or
disturbance in the confluent A549 cell layer. In addition,
our positive control Concanvalin A led to a secretion of
most of the mea sured cytokines and chemokines. From
dead cells, the secretion would not be so pronounced.
T-cell-dependent mediators were measured 72 hours
after adding of the CD4
+
-T-cells , too (see additional file
1, table S1). Although our positive control (Concancava-
lin A) induced a release of most of the mediators (see
additional file 1, table S1), we were not able to measure
a significant release after incubation with SPP. For us,
the most likely explanation of the low concentrations is
the low proportion of MDDC to allergen (Phleumpra-
tense)-specific T-cells. However, it is important to note
thatuptillnowwewereonlyabletocultivatetheT-
cells within the co-culture model for 72 hours in order
to guarantee viability of all cells. Crucial parameters of
the immunological responses within the body normally
occur over longer time periods. This limitation has to
be considered. Thereby, the epithelial airway model
should be further optimized so that a standardized use
of the four different cell types is warranted and T-cell-
dependent mediators and behaviour can be evaluated.
Conclusion
Taken together, SPP uptake in various cell types, i.e.
MDM, EC and MDDC, of a human epithelial airway
model is modulated by SP-D. Thereby, a SPP-induced
inflammation is decreased by SP-D. In addition, the
contact to other lung cells modulates the A549 cells to
react more similar than primary lung cells.
Additional material
Additional file 1: Table S1. Secretion of cytokines and chemokines after
8 hours incubation with subpollen particles (SPP) plus surfactant protein
d (SP-D) as well as further 72 h incubation with fresh medium.
Table 2 Secretion of cytokines and chemokines after 8 hours incubation with subpollen particles (SPP) plus surfactant
protein d (SP-D) and further 72 h incubation with fresh medium in the presence of T-cells
Cells 10 × 10
6
SPP 10 × 10
6
SPP + 1 μg/ml SP-D 10 × 10
6
SPP
+10 μg/ml SP-D
10 μg/ml
SP-D
IL-1 alpha 108.2 ± 22,0 170.6 ± 40.6* 149.6 ± 33.7 192.7 ± 57.4 102.0 ± 25.6
G-CSF 1853 ± 434.3 2823 ± 623.2 2665 ± 566.7 2534 ± 528.2 2596 ± 560.8
TNF-alpha 150.9 ± 43.2 339.4 ± 118.5 250.2 ± 102.4 268.8 ± 103.3 235.3 ± 128.6
MIP-1beta 1211 ± 105.6 1408 ± 143.6* 1297 ± 233,5 1448 ± 183.4 1224 ± 190.0
IL-6 1119 ± 295.9 2327 ± 577.3 1717 ± 284.8 1812 ± 1388.5 1051 ± 151.7
Values are shown as means of at least 8 experiments ± SEM and are given in pg/ml. Detection limit was 3.2 pg/ml. Limit of quantification was: IL-1 alpha 80 pg/
ml; G-CSF 80 pg/ml; TNF alpha 80 pg/ml; MIP-1 beta 80 pg/ml; IL-6 16 pg/ml; * p < 0.05 vs Cells
Schleh et al. Respiratory Research 2012, 13:8
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Page 8 of 10
List of abbreviations
EC: Epithelial Cell; MDM: Monocyte derived Macrophage; MDDC: Monocyte
derived Dendritic Cell; PSG: Pollen Starch Granules; SPP: Subpollen Particle;
SP-D: Surfactat Protein-D
Acknowledgements
The authors thank Bianca Lavae-Mokhtari and Lena Witte for their technical
support. This work was funded by the Swiss National Science Foundation
and the German Research Foundation (SPP 1331) to BR and the German
Research Foundation (SFB 587/B8) to JMH.
Author details
1
Fraunhofer Institute of Toxicology and Experimental Medicine, Nikolai-Fuch-
Str. 1, 30635 Hannover, Germany.
2
MedicalSchoolHannover, Carl-Neuberg-Str.
1, 30625 Hannover, Germany.
3
Respiratory Medicine, Berne University
Hospital, Murtenstrasse 50, Postfach 44, 3010 Bern, Switzerland.
4
Adolphe
Merkle Institute, University of Fribourg, Rte de l’Ancienne Papeterie CP 209,
1723 Marly 1, Switzerland.
5
Technical University Carolo-Wilhelmina
Braunschweig, 38092 Braunschweig Germany.
Authors’ contributions
CS planned the concept and study design, performed the experiments,
interpreted the results and wrote major parts of the manuscript. BMR
planned the concept and study design, interpreted the results and wrote
major parts of the manuscript; VJE planned the concept and study design
and interpreted the results, PG, FB, HDL, MN, AB, and NK made substantial
contributions to the analysis and interpretation of the data. JMH planned
the concept and study design, made substantial contributions to the
analysis and interpretation of the data and wrote major parts of the
manuscript. All of the authors have critically read the manuscript and
approved its submission.
Competing interests
The authors declare that they have no competing interests.
Received: 12 August 2011 Accepted: 1 February 2012
Published: 1 February 2012
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doi:10.1186/1465-9921-13-8
Cite this article as: Schleh et al.: Surfactant Protein D modulates
allergen particle uptake and inflammatory response in a human
epithelial airway model. Respiratory Research 2012 13:8.
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