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Am J Physiol Lung Cell Mol Physiol
281: L1453–L1463, 2001.
Surfactant protein D enhances bacterial antigen
presentation by bone marrow-derived dendritic cells
KAREN G. BRINKER,1 EMILY MARTIN,1 PAUL BORRON,1 ELAHE MOSTAGHEL,2
CAROLYN DOYLE,2 CLIFFORD V. HARDING,3 AND JO RAE WRIGHT1,4
Departments of 1Cell Biology, 2Immunology, and 4Medicine, Duke University
Medical Center, Durham, North Carolina 27710; and 3Institute of Pathology,
Case Western Reserve University, Cleveland, Ohio 44106
Received 13 June 2001; accepted in final form 14 August 2001
antigen presenting cell; innate immune response; adaptive
immune response; phagocytosis; lung
(DCs) are bone marrow-derived antigen
presenting cells (APCs) with the unique capacity to
activate naive T cells and initiate immune responses.
DCs residing in peripheral locations such as the lung
and skin exist in an “immature” form that is capable of
capturing antigen very efficiently by macropinocytosis
(51), endocytosis (51), and phagocytosis (55). Bacterial
and inflammatory products stimulate the maturation
of DCs, resulting in a downregulation of antigen uptake and an upregulation of T cell-stimulatory ability
coincident with DC migration to regional lymph nodes
(5).
DENDRITIC CELLS
Address for reprint requests and other correspondence: J. R.
Wright, Box 3709, Dept. of Cell Biology, Duke Univ. Medical Center,
Durham, NC 27710 (E-mail: [email protected]).
http://www.ajplung.org
DCs are the most potent APC population in the lung,
whereas resident macrophages are ineffective APCs
because they suppress both DC function and T cell
activation (23). DCs in the lung are a rare population of
cells found at all sites of antigen exposure, including
the nasal mucosa, airway epithelium, lung parenchyma, and alveolar surface (37). With an inflammatory stimulus such as bacterial exposure, the number
of DCs at these sites greatly increases (19, 38). Like
other peripheral DCs, DCs isolated from the lung exist
in an immature state in which they are competent for
antigen uptake via receptor-mediated endocytosis but
require additional signals for effective T cell activation
(7, 54). Recently, lung DCs have also been reported to
be phagocytic (16), although the mechanisms of microbial recognition and phagocytosis by lung DCs have not
been investigated.
As sentinels of the immune system, DCs recognize
and present microbial antigens to T cells. DCs have
been shown to phagocytose a broad array of microorganisms (15, 24, 26, 29, 48), resulting in the activation
of antigen-specific T cells (42, 43, 55). Microbial recognition is partly mediated by pattern recognition molecules expressed on DCs, although little is known about
the mechanisms of phagocytic uptake (47). Several
studies (26, 27, 29, 48) have shown that preincubation
of bacteria with serum enhances phagocytosis by DCs,
suggesting that opsonization by serum molecules such
as immunoglobulins or complement factors facilitates
efficient bacterial uptake. Likewise, glycan receptors
may be involved in DC phagocytosis because the uptake of zymosan is inhibited by mannan and ␤-glucan
(48). Other receptors involved in the uptake of particulate antigens by DCs include the fibronectin receptor
(15) and the ␣v␤5-integrin and scavenger receptor
CD36 (1).
Pulmonary surfactant protein (SP) A and SP-D are
pattern recognition molecules that participate in the
innate immune response against bacterial infection
(61). SP-A and SP-D as well as the serum proteins
mannose binding lectin (MBL), conglutinin, and CL-43
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1040-0605/01 $5.00 Copyright © 2001 the American Physiological Society
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Brinker, Karen G., Emily Martin, Paul Borron, Elahe
Mostaghel, Carolyn Doyle, Clifford V. Harding, and Jo
Rae Wright. Surfactant protein D enhances bacterial antigen presentation by bone marrow-derived dendritic cells. Am
J Physiol Lung Cell Mol Physiol 281: L1453–L1463, 2001.—
Surfactant protein (SP) D functions as a soluble pattern
recognition molecule to mediate the clearance of pathogens
by phagocytes in the innate immune response. We hypothesize that SP-D may also interact with dendritic cells, the
most potent antigen presenting cell, to enhance uptake and
presentation of bacterial antigens. Using mouse bone marrow-derived dendritic cells, we show that SP-D binds to
immature dendritic cells in a dose-, carbohydrate-, and calcium-dependent manner, whereas SP-D binding to mature
dendritic cells is reduced. SP-D also binds to Escherichia coli
HB101 and enhances its association with dendritic cells.
Additionally, SP-D enhances the antigen presentation of an
ovalbumin fusion protein expressed in E. coli HB101 to
ovalbumin-specific major histocompatibility complex class II
T cell hybridomas. The enhancement of antigen presentation
by SP-D is dose dependent and is not shared by other collectin-like proteins tested. These studies demonstrate that
SP-D augments antigen presentation by dendritic cells and
suggest that innate immune molecules such as SP-D may
help initiate an adaptive immune response for the purpose of
resolving an infection.
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SP-D ENHANCES DENDRITIC CELL FUNCTION
METHODS
Materials. Media, balanced salt solutions, antibiotics, and
2-mercaptoethanol were purchased from Life Technologies
(Gaithersburg, MD). Fetal bovine serum and heat-inactivated fetal bovine serum were purchased from HyClone (Logan, UT). Agar and yeast extract for Luria broth agar plates
were from Difco Laboratories (Detroit, MI). All other chemicals and reagents were obtained from Sigma (St. Louis, MO)
except where noted.
Antibodies. Rat anti-mouse CD16/CD32 (Fc block), phycoerythrin (PE)- and fluorescein isothiocyanate (FITC)-conjugated mouse anti-mouse I-Ab [major histocompatibility complex (MHC) class II], biotin- or FITC-conjugated rat antiAJP-Lung Cell Mol Physiol • VOL
mouse Ly-6G (GR-1), FITC-conjugated hamster anti-mouse
CD80 (B7-1), FITC- and PE-conjugated rat anti-mouse CD86
(B7-2), PE-conjugated hamster anti-mouse CD11c, and PEconjugated rat anti-mouse CD3 antibodies and all relevant
isotype controls were purchased from PharMingen (San Diego, CA). FITC-conjugated rat anti-mouse macrophage clone
F4180 was obtained from Caltag (Burlingame, CA), and
FITC-conjugated rat anti-mouse CD4 was purchased from
Life Technologies.
Generation of BMDCs. The DC isolation procedure was
adapted from Inaba et al. (25) with some modifications. Bone
marrow cells were flushed from the tibiae and femurs of 6- to
8-wk-old C57BL/6 (H-2b) mice (Charles River Laboratory,
Raleigh, NC) and cultured in 24-well plates at 1 ⫻ 106
cells/ml in DC medium [RPMI 1640 containing 5% heatinactivated fetal bovine serum (FBS), 100 U/ml of penicillinstreptomycin, 20 ␮g/ml of gentamicin, and 50 ␮M 2-mercaptoethanol] in the presence of granulocyte macrophage-colony
stimulating factor [GM-CSF; 5% culture supernatant from
X63 cells transfected with murine GM-CSF cDNA (provided
by Dr. Chris Nicchitta, Duke University, Durham, NC), 20
ng/ml (kindly provided by Immunex, Seattle, WA), or 10
ng/ml (purchased from PharMingen)] for 6 days. The cells
were washed on days 2 and 4 to remove nonadherent cells.
Day 6 nonadherent cells were collected and purified further
by negative selection with biotin-labeled anti-GR-1 followed
by magnetic cell sorting (MACS) streptavidin beads (immature BMDCs; Miltenyi Biotec, Auburn CA). The cells were
matured by reculture in 24-well plates at 3 ⫻ 105 cells/ml in
DC medium in the presence of 100 ng/ml of LPS or, in some
cases, GM-CSF for 24 h. On day 7, the nonadherent cells
were collected and used as mature BMDCs. Immature and
mature cells were characterized for cell surface expression by
flow cytometry. Immature cells were routinely GR-1 negative; CD80, CD86, and MHC class II low; and CD11c positive.
Mature cells were routinely GR-1 negative, MHC class II
high, and CD80, CD86, and CD11c positive.
Generation of OVA-specific T cell hybridomas. Two weeks
before fusion, mice were immunized at the base of tail with
100 ␮g of OVA antigen emulsified in complete Freund’s
adjuvant (CFA). Three days before fusion, draining periaortic
and inguinal lymph nodes were harvested and cells were
restimulated in vitro for 72 h at 37°C in 24-well plates at 4 ⫻
105 lymph node cells 䡠 105 stimulators⫺1 䡠 well⫺1 in a total
volume of 1 ml of complete DMEM. Stimulators were prepared from splenocytes of nonimmunized mice and pulsed for
2 h at 37°C in 10 ml of complete DMEM with 100 ␮g/ml of
OVA258–276 peptide. The pulsed stimulators were then
washed, irradiated (3,000 rads), and resuspended in complete DMEM at 106 cell/ml for plating. On the day of fusion,
in vitro restimulated T cells were washed with DMEM (without FCS or HEPES), counted, and mixed with BW5147 lymphoma cells in log growth at a 1:1 ratio, and fusion was
performed as previously described (8). Ten days after fusion,
the wells were expanded and maintained in 1⫻ hypoxanthine-thymidine medium (Life Technologies) until sufficient
growth had occurred to make duplicate cultures, at which
time the cells were weaned to DMEM with 10% FCS. Hybridomas were screened by fluorescence-activated cell sorting
(FACS) for expression of CD3 and CD4, and only clones
expressing CD3 or CD3 and CD4 were subjected to further
analysis. After screening for antigen specificity, hybridomas
were cloned by limiting dilution (plated at 0.3 cells/well).
Hybridomas were screened for antigen specificity by determination of interleukin (IL)-2 secretion (mouse IL-2 mini-kit,
Endogen, Woburn, MA) in response to a 24-h incubation at
37°C with syngeneic APCs and 100 ␮g/ml of OVA258–276
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are classified as collectins, a family of soluble pattern
recognition molecules named for their NH2-terminal
collagen-like domain and COOH-terminal lectin domain (14). SP-A and SP-D are synthesized and secreted
by alveolar type II and airway Clara cells into the
liquid hypophase that covers the lung. In addition, a
recent study (34) has demonstrated that mRNA for
SP-D has been found in a variety of other mucosal
tissues.
The pulmonary collectins participate in innate immunity with similar yet specialized functions. SP-A
and SP-D act as opsonins to facilitate the removal of
pathogens through interactions of their C-type lectin
domain with carbohydrate and glycolipid structures on
the surface of pathogens (9, 61). Both lung collectins
bind and aggregate several strains of bacteria, viruses,
and allergens (9, 61) and interact with immune cells in
a carbohydrate- and calcium-dependent manner (9,
61). SP-A and SP-D enhance the phagocytosis of bacteria by both alveolar macrophages (49, 56, 60) and
neutrophils (17). The SPs also regulate other immune
functions including chemotaxis, cytokine production,
and T cell proliferation (4, 9, 11, 62). Mice made deficient in SP-A and SP-D by targeted disruption of the
genes are more susceptible to bacterial and viral infection (30–33) and lipopolysaccharide (LPS)-mediated
inflammation (3). Putative receptors for SP-A and
SP-D [SPR-210 (6) and gp-340 (22), respectively] have
been described, although their contributions to many of
the SP-mediated functions have not been confirmed.
The SPs have also been reported to bind to the LPS
receptor CD14 (52).
The goal of the present study was to determine
whether SPs enhance phagocytic uptake and presentation of microbes by DCs. We investigated whether
SP-D was able to interact with a population of immature bone marrow-derived DCs (BMDCs) to enhance
the phagocytosis and presentation of a bacterial model
antigen, Escherichia coli HB101:Crl-OVA (45, 55). We
show that SP-D binds specifically to immature BMDCs, resulting in increased uptake of E. coli HB101
bacteria and enhanced antigen presentation of a defined ovalbumin (OVA) epitope expressed as a fusion
protein in E. coli HB101. Our results indicate that
SP-D augments an adaptive immune response against
bacterial antigens, an important and previously unrecognized function for this innate pattern recognition
molecule.
SP-D ENHANCES DENDRITIC CELL FUNCTION
AJP-Lung Cell Mol Physiol • VOL
200 g, and fixed in 1% paraformaldehyde. Association of
bacteria with the BMDCs was analyzed by FACS and is
reported as “relative fluorescence,” which is defined as the
percent mean fluorescence of all of the cells analyzed in the
presence of added protein compared with the no-protein
control.
Antigen presentation experiments. Antigen presentation
experiments were performed as described by Svensson et al.
(55) with some modifications. E. coli HB101:Crl-OVA bacteria were collected from 16- to 18-h overnight plate cultures
grown on Luria broth-ampicillin and resuspended to 1 ⫻ 109
colony-forming units/ml in PBS as determined by an optical
density at 660 nm reading. Bacteria were added to 2 ⫻ 105
immature BMDCs in a round-bottom 96-well plate at bacteria-to-cell ratios ranging from 5 to 100 in 0.1 ml of Iscove’s
modified Dulbecco’s medium (IMDM) containing 10% heatinactivated FBS. In some cases, OVA protein, synthetic
OVA258–276 peptide, or E. coli HB101 not expressing the
Crl-OVA fusion protein were added to the cells. SP-D and the
other proteins tested were added at the indicated concentrations, and the plates were incubated at 37°C for 3 h. The cells
were washed once with D-PBS, and then antigen uptake and
processing were stopped by fixing the cells with 0.5% paraformaldehyde for 20 min followed by one wash with D-PBS,
incubation for 20 min in D-PBS containing 0.2 M glycine,
four more washes with increasing volumes of D-PBS, and
addition of 0.1 ml of fresh IMDM containing 10% heatinactivated FBS and antibiotics for further incubation. MHC
class II T cell hybridomas specific for OVA258–276 on I-Ab
were then added at 1 ⫻ 105 cells/0.1 ml IMDM containing
10% heat-inactivated FBS and antibiotics. After 24 h, the
supernatants were collected, and an IL-2 ELISA assay was
performed. Negligible IL-2 was produced when T cell hybridomas were incubated with BMDCs that were not exposed to
antigen or when T cell hybridomas were incubated with
OVA258–276 peptide in the absence of prefixed BMDCs. The
IL-2 levels produced varied between experiments probably
due to differences between antigens and expression of the
Crl-OVA protein as well as variation in CD3/CD4 expression
of the T cell hybridomas and variation in the BMDCs from
different cultures. In some experiments, data are reported as
“relative IL-2 production,” which is defined as the percent
IL-2 produced in the presence of protein compared with the
no-protein control.
SP-D-coated bacteria experiments. SP-D was preincubated
with 6 ⫻ 107 E. coli HB101:Crl-OVA in 0.3 ml of FACS buffer
for 30 min at 37°C with shaking in the presence (SP-D-coated
bacteria) and absence (noncoated bacteria) of SP-D at varying concentrations. The bacteria were washed two times and
resuspended in IMDM without antibiotics before addition to
BMDCs. For the appropriate conditions, SP-D was then
added at varying concentrations to the noncoated bacteria.
Cells were incubated for 3 h at 37°C and processed as described in Antigen presentation experiments.
FACS. FACS was performed at the Duke Comprehensive
Cancer Center Flow Cytometry Facility. Samples (⬃10,000
cells/treatment) were analyzed for fluorescence per cell at
514 nm after excitation at 488 nm.
Statistics. Data were compared by analysis of variance
with Tukey’s or Student’s t-test where appropriate. Values
were considered significant at P ⬍ 0.05.
RESULTS
Characterization of BMDCs. DCs isolated after 6
days of culture with GM-CSF consisted predominantly
of immature DCs with a phenotype of CD11c⫹, MHC
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peptide (2 ⫻ 105 responders 䡠 2 ⫻ 105 stimulators⫺1 䡠 well⫺1 in
a total volume of 200 ␮l in 96-well U-bottom plates).
Protein isolation and labeling. Recombinant rat SP-D was
purified by maltose affinity chromatography from the medium supernatant of cultured Chinese hamster ovary cells
expressing a full-length rat SP-D cDNA clone as previously
described (12). The SP-D preparations were tested for endotoxin and were found to have ⬍5 pg endotoxin/␮g SP-D by the
Limulus amebocyte lysate assay QCL-1000 (BioWhittaker,
Walkersville, MD). SP-D was labeled with the Alexa Fluor
488 protein labeling kit (Molecular Probes, Eugene, OR) as
described by the manufacturer, followed by dialysis into
Dulbecco’s PBS (D-PBS) to remove unbound label.
Human SP-A was purified from the bronchoalveolar lavage
fluid of patients with alveolar proteinosis via butanol extraction as previously described (36). SP-A was stored in 5 mM
Tris, pH 7.4, at ⫺20°C. The SP-A preparations were tested
and treated for endotoxin and were found to have ⬍5 pg
endotoxin/␮g SP-A.
MBL was purified from rat serum (Pel-Freeze, Rogers, AR)
by affinity and gel filtration chromatography as previously
described (57).
Human serum complement component 1q (C1q) was purchased from Advanced Research Technologies (San Diego,
CA).
Bacteria. E. coli HB101 were transformed with a construct
encoding an E. coli fusion protein containing the 257–277
epitope of OVA fused to the COOH terminus of crl, an E. coli
gene that encodes a bacterial cytoplasmic protein that regulates curli surface organelle expression as previously described (45). These bacteria, termed HB101:Crl-OVA, were
stored as a 15% glycerol stock at ⫺80°C and grown on Luria
broth agar plates containing 100 ␮g/ml of ampicillin for
16–18 h. Titers of bacteria were used to correlate the optical
density of a bacterial suspension at 660 nm with colonyforming units per milliliter. Expression of the fusion protein
was confirmed by SDS-PAGE and Coomassie blue staining of
bacterial cell lysates. E. coli HB101:Crl-OVA were labeled
with FITC (Molecular Probes, Eugene, OR) as previously
described (49). The bacteria were washed thoroughly to remove excess FITC label and were stored in 15% glycerol at
⫺80°C.
SP-D binding. Immature BMDCs (2 ⫻ 105) or, in some
cases, BMDCs matured for 24 h in the presence of 100 ng/ml
of LPS or GM-CSF were incubated with Alexa 488-labeled
SP-D for 2 h on ice in a final volume of 0.1 ml of FACS buffer
(D-PBS containing 1% BSA). For some studies, Alexa 488labeled SP-D was heat treated at 95°C for 10 min before
addition to the cells. For the carbohydrate inhibition studies,
10 mM maltose, 10 mM fucose, or 10 mM galactosamine were
added at the beginning of the incubation. For calcium-dependent studies, D-PBS containing 0.9 mM calcium chloride or 1
mM EDTA was used. For the dual-binding studies, a PElabeled anti-CD86 antibody was added for the last 30 min of
the incubation. Periodically, the cells were agitated to prevent settling of the cells. After incubation, the cells were
washed two times by centrifugation at 4°C at 200 g and were
then fixed in 1% paraformaldehyde. Binding of SP-D to the
cells was assessed by FACS and is reported as the mean
fluorescence of all the cells analyzed.
Uptake experiments. Immature BMDCs (2 ⫻ 105) plated in
a 96-well plate were incubated with FITC-labeled E. coli
HB101:Crl-OVA at a bacteria-to-cell ratio of 100:1 in the
presence and absence of SP-D (concentrations ranging from
0.5 to 7.5 ␮g/ml) or the indicated concentrations of heattreated SP-D, SP-A, C1q, or MBL for 30 min at 37°C in the
dark. The cells were collected, washed two times at 4°C at
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SP-D ENHANCES DENDRITIC CELL FUNCTION
class IIintermediate, GR-1⫺, CD80low, and CD86low as
described by others (25) (data not shown). After maturation of DCs for 24 h with LPS or GM-CSF, ⬎60% of
BMDCs routinely displayed a mature phenotype characterized by high expression of CD11c, MHC class II,
CD80, and CD86, consistent with expected maturation,
whereas the remaining cells exhibited a less mature
phenotype (8). The phenotype of BMDCs was also assessed by cytospin; mature DCs exhibited a typical
stellate shape (25) (data not shown).
SP-D binds to immature BMDCs. SP-D bound to
immature BMDCs in a calcium-dependent manner
(Fig. 1A), with up to 80% of the cell population positive
for SP-D at 5 ␮g/ml (data not shown). Binding of SP-D
to immature BMDCs increased from 0.1 to 5 ␮g/ml of
SP-D (Fig. 1B). As previously demonstrated with SP-D
binding to leukocytes and epithelial cells (21, 28), binding was completely inhibited by 1 mM EDTA at all
doses tested. Binding was also dependent on the carbohydrate recognition domain of SP-D because binding
was reduced by the addition of excess sugars known to
compete for binding to SP-D (44) as well as by heat
treatment of the protein (Table 1).
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Table 1. Characterization of SP-D binding to BMDCs
Treatment
SP-D Binding, %control
None
Heat-treated SP-D
1 mM EDTA
10 mM maltose
10 mM fucose
10 mM galactosamine
100
16.6 ⫾ 4.83*
15.3 ⫾ 3.72*
13.4 ⫾ 4.99*
31.9 ⫾ 5.07*
94.8 ⫾ 5.79
Values are means ⫾ SE; n ⫽ 3 individual experiments. Binding of
1 ␮g/ml of surfactant protein (SP) D to bone marrow-derived dendritic cells (BMDCs) was performed as in Fig. 1. * P ⬍ 0.005
compared with no treatment.
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Fig. 1. Surfactant protein (SP) D binding to immature bone marrowderived dendritic cells (BMDCs). A: immature BMDCs were incubated with 1 ␮g/ml of Alexa 488-labeled SP-D for 2 h at 4°C in
fluorescence-activated cell sorting (FACS) buffer containing 0.9 mM
CaCl2 or 1 mM EDTA. The cells were washed and fixed for analysis
by FACS, and a representative FACS histogram is shown. B: immature BMDCs were incubated with increasing concentrations of Alexa
488-labeled SP-D, and binding was performed as above. Data are
means ⫾ SE of the mean fluorescence of SP-D binding to the BMDCs;
n ⫽ 3 or 4 individual experiments. Significant difference from EDTA
conditions: * P ⬍ 0.05 by Student’s t-test; # P ⬍ 0.05 by Tukey’s
analysis of variance.
We next compared the binding of SP-D to immature
BMDCs and to BMDCs that were matured by culture
with LPS or GM-CSF for 24 h. Figure 2A shows that
the binding of SP-D to mature BMDCs stimulated with
LPS or GM-CSF was reduced by 31 and 46%, respectively, compared with SP-D binding to immature
BMDCs. To assess whether the reduced binding to the
mature DCs correlated with the maturation state of
the DCs, dual-binding studies were performed with an
anti-CD86 antibody as a maturation marker. As expected, the percentage of cells expressing high levels of
CD86 increased during culture with LPS or GM-CSF.
Interestingly, the level of maturation induced by LPS
and GM-CSF differed, although both stimuli induced
the differentiation of cells expressing a high level of
CD86. As indicated by FACS analysis, SP-D bound to
the majority of immature, CD86low BMDCs, whereas
the proportion of cells low for SP-D binding increased
with increasing expression of CD86 (Fig. 2B). Compared with binding to immature cells, the mean fluorescence of the SP-D binding was reduced on all populations of BMDCs matured with either LPS or GMCSF, with the most marked decrease observed on the
most mature, high CD86-expressing cells. Similar results were seen with MHC class II as a maturation
marker, although the data were not as clear-cut as
with CD86 because the populations of low, intermediate, and high MHC class II cells were not as easily
distinguishable as when CD86 was used as a maturation marker, particularly in experiments where overall
maturation was lower (data not shown). Samples incubated with a single label were analyzed to exclude the
effect of spectral overlap between the FITC and PE
channels and to ensure that there was no interaction
between the Alexa-labeled SP-D and the PE anti-CD86
antibody during the binding incubation (data not
shown). Indeed, there was no difference in SP-D binding to the immature or mature BMDCs in the presence
and absence of the dual label (data not shown).
Our initial studies employed a positive selection
strategy for the isolation of DCs from GM-CSF-cultured mouse bone marrow cells with Miltenyi magnetic
microbeads (MACS) against CD11c. However, we discovered that SP-D bound to the MACS beads used in
the isolation. All of the studies reported here were done
with cells purified by MACS-negative selection so that
SP-D ENHANCES DENDRITIC CELL FUNCTION
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Fig. 2. SP-D binding to immature and mature BMDCs. A: immature or
mature BMDCs [matured with lipopolysaccharide (LPS) or granulocyte-macrophage colony-stimulated factor (GM-CSF)] were incubated
with 2.5 ␮g/ml of Alexa 488-labeled SP-D for 2 h at 4°C in FACS buffer.
The cells were washed and fixed for analysis by FACS. Data are
means ⫾ SE of the percentage of cells positive for SP-D binding to the
BMDCs; n ⫽ 3 or 4 individual experiments. *P ⬍ 0.05 compared with
immature BMDCs by Student’s t-test. B: for the last 30 min, a phycoerythrin (PE) anti-CD86 antibody was added. The cells were washed
and fixed for analysis by dual FACS. Data are means ⫾ SE of representative FACS plots with mean fluorescence values included; n ⫽ 3
individual experiments for arbitrarily gated low (lo), intermediate (int),
and high (hi) CD86-expressing cell populations.
the cells used did not have MACS beads. Importantly,
although the trends obtained with both positively and
negatively selected cells were similar, the presence of
the beads affected the level of SP-D binding to BMDCs
AJP-Lung Cell Mol Physiol • VOL
Fig. 3. SP-D enhancement of Escherichia coli HB101:Crl-OVA association with immature BMDCs. A: immature BMDCs were incubated
with FITC-labeled E. coli HB101:Crl-OVA in the presence and absence of 5 ␮g/ml of SP-D for 30 min at 37°C. BMDCs were collected,
washed, and fixed for analysis by FACS. A representative FACS plot
is shown. B: association experiments were performed as above in the
presence and absence of increasing concentrations of SP-D. Data are
means ⫾ SE; n ⫽ 6 individual experiments. * P ⬍ 0.05 compared with
no-protein condition.
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as well as the magnitude of the SP-D-mediated effect
on the uptake and presentation of bacterial antigens.
SP-D enhances the association of E. coli HB101 with
immature BMDCs. SP-D has previously been shown
(17, 49) to enhance the phagocytosis of gram-negative
bacteria by both alveolar macrophages and neutrophils. Therefore, we tested the ability of SP-D to enhance the phagocytosis of the E. coli strain HB101 by
BMDCs. SP-D enhanced the association of E. coli
HB101 with BMDCs after a 30-min incubation with
BMDCs and FITC-labeled E. coli HB101 (Fig. 3A). As
evidenced in the FACS plot in Fig. 3A, the effect of
SP-D on bacterial association resulted in an increase in
the fluorescence of the BMDCs rather than in an increase in the number of cells that were positive for the
labeled bacteria, suggesting that SP-D mediates bacterial aggregation of E. coli HB101. Indeed, as assessed
by a spectrophotometric aggregation assay, SP-D aggregated E. coli HB101 (data not shown). The enhancement by SP-D on bacterial association increased with
increasing doses of SP-D from 0.5 to 5 ␮g/ml, at which
point the enhancement by SP-D reached a plateau (Fig.
3B). Unfortunately, FITC-labeled E. coli HB101 could
not be quenched by trypan blue (50), and thus the data
presented here depict total bacterial association with
the BMDCs, including both binding and uptake of the
bacteria to the BMDCs. Analysis of the cells by confocal
microscopy confirmed that bacteria were indeed internalized (data not shown).
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SP-D ENHANCES DENDRITIC CELL FUNCTION
SP-D enhances the antigen presentation of E. coli
HB101:Crl-OVA. E. coli HB101 expressing a fusion
protein containing the 257–277 epitope of OVA
(HB101:Crl-OVA) was used to assess the antigen presentation of the immunodominant OVA258–276 epitope
by BMDCs to an MHC class II-restricted T cell hybridoma. Because immature, but not mature, DCs are
required to phagocytose particulate antigens for antigen presentation (1), immature BMDCs were incubated with E. coli HB101:Crl-OVA followed by fixation
and several washes before the addition of T cell hybridomas. Figure 4A shows that in the absence of SP-D, the
Crl-OVA antigen is presented in a dose-dependent
manner, with IL-2 produced after exposure to as few as
1 ⫻ 106 bacteria (a ratio of 5 bacteria to 1 BMDC). IL-2
production began to plateau from 1–2 ⫻ 107 total
bacteria (50–100 bacteria to BMDCs). This was not
due to changes in the viability of the BMDCs in the
presence of increasing doses of bacteria (data not
shown). In the presence of 1 ␮g/ml of SP-D, there was
Fig. 5. SP-D dose-dependent enhancement of antigen presentation
of E. coli HB101:Crl-OVA. A: immature BMDCs were incubated with
E. coli HB101:Crl-OVA (100:1 bacteria to BMDCs) in the presence
and absence of increasing concentrations of SP-D for 3 h at 37°C. The
cells were fixed and washed, and OVA258–276/I-Ab specific T cell
hybridomas were added for 24 h. The supernatants were collected,
and an IL-2 ELISA was performed. Data are means ⫾ SE; n ⫽ 3
individual experiments. * P ⬍ 0.05 compared with no-SP-D condition.
AJP-Lung Cell Mol Physiol • VOL
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Fig. 4. SP-D enhancement of antigen presentation of E. coli HB101:
Crl-OVA. A: immature BMDCs were incubated with increasing concentrations of E. coli HB101:Crl-OVA ([bacteria]) in the presence and
absence (⫺) of 1 ␮g/ml of SP-D for 3 h at 37°C. The cells were fixed
and washed, and OVA258–276/I-Ab T cell hybridomas were added for
24 h. The supernatants were collected, and an interleukin (IL)-2
ELISA was performed. Data are from a representative experiment.
B: experiments were performed as described in A. [IL-2], IL-2 concentration. Data are means ⫾ SE and summarize all experiments;
n ⫽ 3 individual experiments. * P ⬍ 0.05 compared with no-SP-D
condition by Student’s t-test.
a consistent enhancement of the antigen presentation
of E. coli HB101:Crl-OVA relative to the antigen presentation in the absence of SP-D at all bacterial concentrations (Fig. 4A). The percent increase in IL-2
production due to the presence of SP-D at all bacterial
concentrations is depicted in Fig. 4B.
As seen in Fig. 5, SP-D enhanced the antigen presentation of E. coli HB101:Crl-OVA in a dose-dependent manner, with significant enhancement observed
with as low as 0.1 ␮g/ml of SP-D. The enhancement of
antigen presentation increased with increasing doses
of SP-D from 0.025 to 1 ␮g/ml, at which point the
enhancement by SP-D was maximal.
Incubation of DCs with E. coli HB101 not expressing
the Crl-OVA construct did not result in antigen presentation as measured by IL-2 production (data not
shown). Furthermore, there was virtually no presentation of E. coli HB101:Crl-OVA by mature DCs under all
conditions tested (data not shown).
Enhancement of antigen presentation of E. coli HB101:
Crl-OVA is specific to SP-D. To examine whether the
enhancement of E. coli HB101:Crl-OVA association
and presentation was specific to SP-D, other members
of the collectin family and the structurally homologous
C1q were tested in experiments with BMDCs. Table 2
shows that, of the proteins tested, only SP-D significantly enhanced both bacterial association and antigen
presentation, whereas enhancement was reduced by
heat treatment of the protein. Interestingly, SP-A,
C1q, and MBL significantly enhanced the association
of E. coli HB101 with BMDCs to levels that were
similar to the enhancement seen with SP-D. However,
unlike SP-D, the other collectin-like proteins tested did
not affect the antigen presentation of E. coli HB101:
Crl-OVA at any of the protein concentrations tested.
SP-D does not enhance the antigen presentation of
OVA protein or peptide. To investigate whether the
SP-D-mediated enhancement of antigen presentation
was a result of a direct effect of SP-D on the BMDCs,
SP-D ENHANCES DENDRITIC CELL FUNCTION
L1459
Table 2. Effects of collectin-like proteins
on the association and antigen presentation
of HB101:Crl-OVA by BMDCs
Proteins Tested
Antigen Presentation,
relative IL-2 production
100
100
123 ⫾ 21
283 ⫾ 41*
237 ⫾ 25*
227 ⫾ 23*
105 ⫾ 20
130 ⫹ 34
135 ⫾ 8
ND
ND
178 ⫾ 24*
ND
93 ⫾ 11
104 ⫾ 10
94 ⫾ 14
ND
190 ⫾ 37*
ND
86 ⫾ 7
84 ⫾ 7
117 ⫾ 7
ND
210 ⫾ 38*
86 ⫾ 4
106 ⫾ 3
Values are means ⫾ SE; n ⫽ 5–6 individual experiments for
association and 3–6 individual experiments for antigen presentation. OVA, ovalbumin; IL, interleukin; ht SP-D, heat-treated SP-D;
C1q, complement component 1q; MBL, mannose binding lectin; ND,
not determined. Association experiments are described in Fig. 3.
Antigen presentation experiments are described in Fig. 4. * P ⬍ 0.05
compared with no protein.
OVA protein or peptide was used in antigen presentation experiments. The data show that both OVA protein and peptide were presented by BMDCs to MHC
class II T cell hybridomas in a dose-dependent manner.
However, SP-D did not enhance the antigen presentation of OVA at any of the doses tested (Fig. 6A), in
contrast to its effects on E. coli HB101:Crl-OVA presentation. Likewise, SP-D did not enhance the antigen
presentation of the OVA258–276 peptide and statistically inhibited antigen presentation at 25 ␮g/ml of
peptide (Fig. 6B). It is not clear whether this inhibition
may have been due to SP-D binding to the peptide.
Thus the SP-D-mediated enhancement of antigen presentation of E. coli HB101:Crl-OVA does not appear to
be a direct effect of SP-D on BMDCs but may be due to
its interaction with E. coli HB101.
SP-D binds E. coli HB101:Crl-OVA and enhances
antigen presentation. Both SP-A and SP-D have been
shown to bind various pathogens to mediate phagocytosis by immune cells (17, 46). The ability of SP-D to
bind E. coli HB101 was tested by precoating the bacteria with SP-D and removing unbound SP-D before
addition to the BMDCs. The data show that SP-D
enhanced the antigen presentation of E. coli HB101:
Crl-OVA with BMDCs to a greater extent when the
bacteria were first coated with SP-D (Fig. 7A). The
enhancement by SP-D-coated bacteria on antigen presentation averaged 529 ⫾ 178.9% at 1 ␮g/ml of SP-D
compared with the no-protein control (data not shown)
and was significantly greater than noncoated E. coli
HB101 at SP-D concentrations of 0.1 ␮g/ml and greater
as assessed by a paired Student’s t-test (P ⬍ 0.05; data
not shown).
AJP-Lung Cell Mol Physiol • VOL
Fig. 6. SP-D does not enhance the antigen presentation of OVA
protein or peptide. A: immature BMDCs were incubated with increasing concentrations of OVA protein ([OVA]) in the presence and
absence of 1 ␮g/ml of SP-D for 3 h at 37°C. The cells were fixed and
washed, and OVA258–276/I-Ab specific T cell hybridomas were added
for 24 h. The supernatants were collected, and an IL-2 ELISA was
performed. Data are means ⫾ SE; n ⫽ 3 individual experiments. B:
experiments were performed and analyzed as in A except with
OVA258–276 peptide. Data are means ⫾ SE; n ⫽ 3 individual experiments. * P ⬍ 0.05 compared with no-SP-D condition by Student’s
t-test.
DISCUSSION
This study demonstrates that SP-D is able to interact with APCs to enhance the phagocytosis and antigen
presentation of bacteria. We show that SP-D binds to a
Fig. 7. SP-D binds E. coli HB101:Crl-OVA and enhances antigen
presentation. E. coli HB101:Crl-OVA were coated or not coated with
increasing concentrations of SP-D from 0.05 to 1 ␮g/ml and added to
immature BMDCs for 3 h at 37°C. After overnight culture with
OVA258–276/I-Ab specific T cell hybridomas, the supernatants were
collected, and an IL-2 ELISA was performed. Data are from a
representative experiment.
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None
SP-D
1 ␮g/ml
5 ␮g/ml
ht SP-D
1 ␮g/ml
5 ␮g/ml
SP-A
1 ␮g/ml
25 ␮g/ml
50 ␮g/ml
C1q
1 ␮g/ml
25 ␮g/ml
50 ␮g/ml
MBL
1 ␮g/ml
25 ␮g/ml
Association,
relative fluorescence
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SP-D ENHANCES DENDRITIC CELL FUNCTION
AJP-Lung Cell Mol Physiol • VOL
hance the antigen presentation of the OVA protein or a
synthetic OVA258–276 peptide, suggesting that SP-D is
not able to directly regulate components of the antigen
processing and presentation pathway such as MHC
class II peptide loading, MHC class II turnover, costimulatory molecules, or cytokines that would lead to
an increased activation of T cells. However, we cannot
exclude the possibility that SP-D may elicit changes in
cell surface molecules or cytokine production that
would affect T cell function under different experimental conditions such as longer incubation times. Furthermore, the T cell hybridomas used in this study are
insensitive to the requirements of costimulation (8),
and it is possible that SP-D may regulate the expression of some molecules required for T cell activation in
other systems. The ability of SP-D to stimulate the
production of cytokines and expression of cell surface
molecules by DCs will be the focus of future studies.
Second, preincubation of E. coli HB101:Crl-OVA
with SP-D before addition to the BMDCs resulted in
significantly greater enhancement of antigen presentation compared with the condition in which SP-D was
added to the BMDCs at the same time as the bacteria.
This suggests that SP-D-coated E. coli HB101 in the
absence of free SP-D is sufficient to enhance antigen
uptake and presentation. The ability of SP-D to bind
and enhance the phagocytosis of pathogens by immune
cells has been previously reported (reviewed in Ref.
10), although the mechanism for the enhanced uptake
differs between microorganisms. Hartshorn et al. (18)
have reported that SP-D binds and aggregates influenza A virus, resulting in enhanced uptake of the virus
by neutrophils independent of a cellular receptor for
SP-D. In contrast, a study by Restrepo et al. (49)
demonstrated that SP-D acts as an opsonin to enhance
the uptake of Pseudomonas aeruginosa by alveolar
macrophages. Despite the binding of SP-D to immature
DCs, the present study cannot distinguish between a
SP-D receptor-dependent or -independent mechanism
for the enhanced uptake and presentation of E. coli
HB101:Crl-OVA because both SP-D binding to the cell
and aggregation of bacteria are calcium and carbohydrate dependent.
Interestingly, all of the collectin-like proteins tested
enhanced the association of E. coli HB101 with immature DCs, yet only SP-D stimulated significant enhancement of antigen presentation. An intriguing possible mechanism for the specific enhancement of
antigen presentation by SP-D is that SP-D-mediated
uptake of E. coli HB101 targets the bacteria to the
antigen processing and presentation pathway, whereas
the other proteins direct the uptake of the bacteria into
an alternate pathway of the cell. Although little is
known about the intracellular trafficking of SP-A and
SP-D after cellular uptake, it is likely that differences
in the cell surface receptors for these proteins may
govern their traffic inside the cell. A study (53) has
shown that enhanced antigen presentation by the B
cell receptor and Fc receptor is linked to the existence
of sequence motifs in the cytoplasmic domains of the
receptors. These specialized motifs specify compart-
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population of immature BMDCs in a dose-, calcium-,
and carbohydrate-dependent manner. Furthermore,
SP-D binds to E. coli HB101 and augments its association with DCs, resulting in a dose-dependent enhancement of antigen presentation of an OVA fusion
protein expressed in E. coli HB101. Although SP-D has
previously been shown to enhance the phagocytosis of
bacteria by immune cells (61), this is the first study to
demonstrate a linkage between the SP-D-mediated enhancement of phagocytosis and the resultant presentation of antigen, thus showing that SP-D is an important molecule in both the innate and adaptive immune
responses.
The binding of SP-D to immature BMDCs and its
reduced binding to mature BMDCs, identified by their
increased expression of CD86, are consistent with the
antigen-capturing functions of immature DCs and the
finding that other pattern recognition molecules are
also expressed at higher levels on immature than on
mature DCs. For example, the mannose receptor is
highly expressed on immature DCs where it is important in enhancing the internalization and antigen presentation of mannosylated antigens, whereas its expression on mature DCs is reduced by ⬃50% (13, 51).
Similarly, the Toll-like receptor-3 and scavenger receptor CD36 are more highly expressed on immature DCs
than on cells matured in the presence of bacterial
products or cytokines (1, 40). gp-340 and CD14 have
been identified as putative SP-D receptors (22, 52).
Although the expression and regulation of gp-340 on
DCs are not known, it is unlikely that CD14 is the
SP-D receptor on DCs because this molecule has been
shown to be upregulated on DCs matured with LPS
(35).
Our studies demonstrate that SP-D increases T cell
IL-2 production at a particular bacterial concentration
by approximately fivefold when the bacteria are first
coated with SP-D before addition to the BMDCs. Likewise, the efficiency of bacterial antigen presentation by
BMDCs is increased in the presence of SP-D because
fewer bacteria are required to achieve comparable antigen presentation compared with bacteria incubated
in the absence of SP-D. This magnitude of enhancement of bacterial uptake and presentation by SP-D is
similar to the reported effects of SP-D on the phagocytosis of viruses and bacteria by alveolar macrophages
and neutrophils (17, 49). The effect is also consistent
with another study (48) demonstrating that soluble
molecules in serum enhance bacterial uptake by Langerhans cells approximately twofold. Recently, it has
been demonstrated that SP-D is a physiologically important molecule in the immune response because
SP-D-deficient mice are more susceptible to bacterial
infection (33). Future studies with mice lacking SP-D
may provide insight into the importance of SP-D in the
initiation of an adaptive immune response against
bacterial infection.
Several lines of evidence indicate that the ability of
SP-D to enhance the antigen uptake and presentation
of E. coli HB101:Crl-OVA may be due to its direct
interaction with bacteria. First, SP-D is unable to en-
SP-D ENHANCES DENDRITIC CELL FUNCTION
AJP-Lung Cell Mol Physiol • VOL
lation of immune responses by the local milieu of the
lung.
Previous studies (4, 59) have shown that SP-D inhibits the proliferation of T cells stimulated with mitogens. These studies and the present study considered
together are consistent with the possibility that SP-D
is able to both initiate an immune response and control
inflammation against pathogens through the compartmentalization of the immune response. SP-D is found
in the alveolar space of the lung where we postulate it
is able to interact with immature DCs to enhance
antigen uptake and processing and to interact with T
cells to prevent their activation and protect the fragile
lung epithelium against damaging inflammatory products. After antigen uptake, DCs mature and migrate to
regional lymph nodes to activate T cells and initiate an
immune response (20). Thus the initiation of the immune response occurs in regional lymph nodes where
no detectable mRNA has been found for SP-D (2). The
studies described here address the ability of SP-D to
interact with immature DCs, as may occur in the
alveolar space, but do not address the interaction of
SP-D with T cells because SP-D was incubated with the
BMDCs in the antigen presentation experiments followed by fixation and several washes before the addition of the T cell hybridomas. The SP-D-mediated control of a compartmentalized immune response is
intriguing and deserves further study.
In summary, these data identify a new role for SP-D
in recognizing nonself-pathogens and initiating a protective immune response for their removal. It is tempting to speculate that the function of SP-D is not limited
solely to E. coli HB101 and that SP-D may be able to
enhance the antigen presentation of a broad range of
pathogens including bacteria, viruses, and allergens
that it recognizes as nonself. Likewise, the finding that
message for SP-D exists in many mucosal tissues suggests that SP-D may function as a universal pattern
recognition molecule able to regulate the immune response at all sites of pathogen invasion. A broader
understanding of the role of SP-D in activating and
regulating the adaptive immune response may provide
a basis for the use of SP-D as a therapeutic agent to
elicit an efficient immune response against a defined
antigen.
We thank members of the laboratory of Dr. Eli Gilboa (Department of Surgery, Duke University, Durham, NC) for technical assistance regarding the mouse bone marrow-derived dendritic cell culture and isolation. We are grateful to Immunex for kindly providing
us with the recombinant murine granulocyte-macrophage colonystimulating factor used in these studies. We thank Eric Walsh,
Hollie Garner, Patty Keating, and Joel Herbein for technical assistance in the isolation of recombinant surfactant protein D, human
surfactant protein A, and rat mannose binding lectin. We thank Dr.
Mike Cook and Lynn Martinek for technical assistance and analysis
of data by fluorescence-activated cell sorting at the Duke Comprehensive Cancer Center Flow Cytometry Facility. We also express
gratitude to Matthew Potter for critical review of the manuscript.
This work was supported by the National Heart, Lung, and Blood
Institute Grant RO1-HL-51134 and National Institute of General
Medical Sciences Cell and Molecular Biology Training Grant 5-T32GM-07184.
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ment localization of receptor-antigen complexes within
the endocytic pathway through interactions with adaptor proteins and also initiate signaling cascades, resulting in accelerated movement to the antigen processing compartment. Likewise, it is possible that SP-A
and SP-D may differentially regulate or interact with
other components of the trafficking machinery.
However, there are a number of other reasons that
may be equally plausible in explaining the lack of
correlation between protein-mediated enhancement of
bacterial association and enhancement of antigen presentation. First, in the association experiments, SP-A,
C1q, and MBL enhanced the number of cells that were
positive for labeled bacteria, whereas SP-D enhanced
the number of bacteria associated with each positive
cell as evidenced by the increase in the fluorescence of
the positive cells. This finding supports the idea that
SP-D is mediating the aggregation of E. coli HB101.
Bacterial aggregation may result in enhanced antigen
presentation by concentrating the bacteria in the antigen processing pathway after uptake by macropinocytosis or receptor-mediated endocytosis. Second, although experiments with the OVA peptide and protein
suggest that SP-D is not maturing the DCs, it is possible that the interaction of SP-D with the bacteria
alters its ability to activate host cells. This hypothesis
is consistent with a recent study by Ofek et al. (41)
demonstrating that SP-D enhances LPS-induced nitric
oxide production in the presence of particulate LPS or
bacteria but not in the presence of soluble LPS (41).
Third, labeling E. coli HB101 with FITC may have
altered its interaction with the proteins in the association experiments compared with the antigen presentation experiments where unlabeled E. coli HB101 was
used. Thus the association data may not accurately
reproduce the uptake of the bacteria in the antigen
presentation experiments. Finally, based on previous
studies reviewed in Ref. 14 demonstrating that different collectins bind to and enhance phagocytosis of
specific pathogens, we conjecture that the collectins
may differentially function to enhance particulate antigen presentation and propose that studies on other
pathogens may provide insight into the involvement of
the other collectins in antigen presentation.
BMDCs were used in these studies due to the difficulty in purifying lung DCs. The in vitro culture systems used to generate BMDCs have been well described (25) and are useful because they allow the
generation of large numbers of cells with very defined
phenotypes for study. Likewise, a study (7) has shown
that DCs freshly isolated from the lung share many of
the same characteristics as those cultured in vitro.
However, we cannot exclude the possibility that the
interactions of SPs with lung DCs may differ compared
with DCs derived from the bone marrow. Recent improvements have been made in the techniques used for
isolating lung DCs such that it is possible to obtain
pure populations of immature DCs without activating
the cells during the isolation procedure (54, 58). Thus
future studies specifically on the interaction of surfactant with lung DCs may provide insight into the regu-
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SP-D ENHANCES DENDRITIC CELL FUNCTION
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