Soluble CD14 and CD83 from human neonatal antigen-presenting cells are inducible by commensal bacteria and suppress allergen-induced human neonatal Th2 differentiation.
ABSTRACT CD14 is expressed on the cell surface of various antigen-presenting cells, and CD83 is a maturation marker for dendritic cells (DC). CD14 and CD83 are also present as soluble proteins, and both have immunoregulatory functions. We examined whether neonatal cord blood monocytes or DC released soluble CD14 (sCD14) or sCD83 when exposed to the commensal intestinal bacteria Clostridium perfringens, Staphylococcus aureus, Lactobacillus rhamnosus, Escherichia coli, and Bacteroides fragilis. We found that the gram-positive bacteria C. perfringens and S. aureus, but not gram-negative bacteria, induced the release of sCD14 from monocytes. DC, on the other hand, released sCD14 in response to both gram-positive and gram-negative bacteria. Moreover, the expression of the virulence factor staphylococcal protein A seemed to be important for S. aureus-induced sCD14 production from both monocytes and DC. Soluble CD83 was released from DC, but not from monocytes, when exposed to both gram-positive and gram-negative bacteria. Finally, to investigate whether sCD14 or sCD83 could modulate neonatal allergen-induced T-cell differentiation, DC were exposed to birch allergen alone or in the presence of sCD14 or sCD83 and then cocultured with autologous T cells. We demonstrate that sCD14 and sCD83 inhibited the birch allergen-induced Th2 differentiation by suppressing interleukin 13 production. Together, these results suggest that the commensal intestinal flora may be an important stimulus for the developing immune system by inducing the immunoregulatory proteins sCD14 and sCD83, which may be involved in preventing T-cell sensitization to allergens in infants.
- SourceAvailable from: Matthias Lechmann[show abstract] [hide abstract]
ABSTRACT: CD83 is up-regulated on the surface of dendritic cells (DCs) during maturation and has been widely used as a marker for mature DCs. Recently, we reported the recombinant expression of the extracellular immunoglobulin domain of human CD83 (hCD83ext). Using this soluble form of CD83, allogeneic as well as specific cytotoxic T lymphocyte proliferation could be blocked in vitro. Here we report the functional analysis of soluble CD83 in vivo, using murine experimental autoimmune encephalomyelitis (EAE) as a model. Strikingly, only three injections of soluble CD83 prevented the paralysis associated with EAE almost completely. In addition, even when the EAE was induced a second time, CD83-treated mice were protected, indicating a long-lasting suppressive effect. Furthermore, soluble CD83 strongly reduced the paralysis in different therapeutic settings. Most important, even when the treatment was delayed until the disease symptoms were fully established, soluble CD83 clearly reduced the paralyses. In addition, also when EAE was induced a second time, soluble CD83-treated animals showed reduced disease symptoms. Finally, hCD83ext treatment almost completely reduced leukocyte infiltration in the brain and in the spinal cord. In summary, this work strongly supports an immunosuppressive role of soluble CD83, thereby indicating its therapeutic potential in the regulation of immune disorders in vivo.Journal of Experimental Medicine 09/2004; 200(3):345-51. · 13.21 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The intestinal microflora is a likely source for the induction of immune deviation in infancy. The purpose of this study was to prospectively relate the intestinal microflora to allergy development in 2 countries differing with respect to the prevalence of atopic diseases. Newborn infants were followed prospectively through the first 2 years of life in Estonia (n = 24) and Sweden (n = 20). By that age, 9 Estonian and 9 Swedish infants had developed atopic dermatitis and/or positive skin prick test results. Stool samples were obtained at 5 to 6 days and at 1, 3, 6, and 12 months, and 13 groups of aerobic and anaerobic microorganisms were cultivated through use of standard methods. In comparison with healthy infants, babies who developed allergy were less often colonized with enterococci during the first month of life (72% vs 96%; P <.05) and with bifidobacteria during the first year of life (17% to 39% vs 42% to 69%; P <.05). Furthermore, allergic infants had higher counts of clostridia at 3 months (median value, 10.3 vs 7.2 log(10); P <.05). The prevalence of colonization with Staphylococcus aureus was also higher at 6 months (61% vs 23%; P <.05), whereas the counts of Bacteroides were lower at 12 months (9.9 vs 10.6 log(10); P <.05). Differences in the composition of the gut flora between infants who will and infants who will not develop allergy are demonstrable before the development of any clinical manifestations of atopy. Because the observations were made in 2 countries with different standards of living, we believe that our findings could indicate a role for the intestinal microflora in the development of and protection from allergy.Journal of Allergy and Clinical Immunology 10/2001; 108(4):516-20. · 12.05 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The effect of the digestive microflora on oral tolerance to ovalbumin was studied by using axenic (germfree) and conventional C3H/HeJ mice. In contrast to reported results of studies with sheep erythrocytes, oral administration of ovalbumin induced tolerance in axenic mice, but the maintenance of tolerance was found to be of shorter duration than was with conventional mice. These data indicate that the contribution of the microflora to oral tolerance depends on the antigen used.Infection and Immunity 11/1988; 56(10):2766-8. · 4.07 Impact Factor
INFECTION AND IMMUNITY, Aug. 2007, p. 4097–4104
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 75, No. 8
Soluble CD14 and CD83 from Human Neonatal Antigen-Presenting
Cells Are Inducible by Commensal Bacteria and Suppress
Allergen-Induced Human Neonatal Th2 Differentiation?†
Anna-Carin Lundell,1* Kerstin Andersson,1Elisabet Josefsson,1
Alexander Steinkasserer,2and Anna Rudin1
Department of Rheumatology and Inflammation Research, The Sahlgrenska Academy at Go ¨teborg University,
Go ¨teborg, Sweden,1and Department of Dermatology, University Hospital Erlangen, Erlangen, Germany2
Received 1 November 2006/Returned for modification 8 December 2006/Accepted 21 May 2007
CD14 is expressed on the cell surface of various antigen-presenting cells, and CD83 is a maturation marker
for dendritic cells (DC). CD14 and CD83 are also present as soluble proteins, and both have immunoregulatory
functions. We examined whether neonatal cord blood monocytes or DC released soluble CD14 (sCD14) or
sCD83 when exposed to the commensal intestinal bacteria Clostridium perfringens, Staphylococcus aureus,
Lactobacillus rhamnosus, Escherichia coli, and Bacteroides fragilis. We found that the gram-positive bacteria C.
perfringens and S. aureus, but not gram-negative bacteria, induced the release of sCD14 from monocytes. DC,
on the other hand, released sCD14 in response to both gram-positive and gram-negative bacteria. Moreover,
the expression of the virulence factor staphylococcal protein A seemed to be important for S. aureus-induced
sCD14 production from both monocytes and DC. Soluble CD83 was released from DC, but not from monocytes,
when exposed to both gram-positive and gram-negative bacteria. Finally, to investigate whether sCD14 or
sCD83 could modulate neonatal allergen-induced T-cell differentiation, DC were exposed to birch allergen
alone or in the presence of sCD14 or sCD83 and then cocultured with autologous T cells. We demonstrate that
sCD14 and sCD83 inhibited the birch allergen-induced Th2 differentiation by suppressing interleukin 13
production. Together, these results suggest that the commensal intestinal flora may be an important stimulus
for the developing immune system by inducing the immunoregulatory proteins sCD14 and sCD83, which may
be involved in preventing T-cell sensitization to allergens in infants.
Allergic diseases are characterized by a Th2-type immune
response, resulting in immunoglobulin E (IgE) production
against innocuous environmental allergens. It has been sug-
gested that a lack of exposure to a wide variety of microbes
during early childhood may result in a higher prevalence of
allergic diseases in the Western world than in developing coun-
tries (12, 28). This altered microbial exposure affects the nor-
mal gastrointestinal microflora, which may be important for
appropriate development of the immune system (33, 35). Al-
though the immunological mechanisms behind the protection
against allergy are not clear, innate immune cells are most
likely involved, as the first interaction between bacteria and the
immune system occurs via these cells.
Bacterial structures are recognized by pattern recognition
receptors, such as CD14 and Toll-like receptors (TLRs) (1),
which thereby represent a link between the innate and adaptive
immune systems. Traditionally, CD14 has been a hallmark of
monocytes and macrophages, as most subpopulations of these
cells express CD14 (42). In blood, however, dendritic cell (DC)
preparations contain several phenotypically and functionally
distinct subpopulations, of which some express low levels of
CD14 (31). CD14 plays a key role in initiating cell activation by
a group of bacterially derived structures, such as lipopolysac-
charide (LPS) from gram-negative bacteria and peptidoglycan
from gram-positive and gram-negative bacteria (15, 39). A
soluble form of CD14 (sCD14) is present in large amounts in
human serum and in breast milk (14, 26) and may be an
acute-phase protein with the function of protecting against
LPS-induced shock (7, 21). In addition, sCD14 has immuno-
regulatory functions, as it interacts with activated human T and
B cells, leading to inhibition of interleukin 4 (IL-4), gamma
interferon (IFN-?), and IgE production, respectively (5, 34).
Interestingly, it was recently shown that the circulating levels of
sCD14 were reduced in 7-year-old atopic children compared
with nonatopic children (41), and house dust mite-sensitized
children had significantly lower levels of sCD14 in the circula-
tion than those not sensitized to house dust mites (36). More-
over, we have recently shown that children who harbor Staph-
ylococcus aureus in the gut early in life have higher levels of
sCD14 in the circulation than do noncolonized children and
that children who developed food allergies tended to have
lower levels of sCD14 in plasma (30).
Human CD83 is a 45-kDa glycoprotein and a member of the
immunoglobulin superfamily, which has been widely used as
one of the best surface markers for activated DC (6). Soluble
CD83 (sCD83) is released from activated in vitro-cultured
human DC and is detectable in small amounts in the circula-
tion of healthy infants and adult individuals (19, 30). Soluble
CD83 has been shown to regulate immune responses by inhib-
iting DC–T-cell clustering and DC-mediated T-cell expansion
* Corresponding author. Mailing address: Department of Rheuma-
tology and Inflammation Research, Go ¨teborg University, Guldheds-
gatan 10, 413 46 Go ¨teborg, Sweden. Phone: 46-31-342 64 11. Fax:
46-31-82 39 25. E-mail: email@example.com.
† Supplemental material for this article may be found at http://iai
?Published ahead of print on 25 May 2007.
(25, 29). Moreover, administration of sCD83 has been found to
ameliorate Th1-driven experimental autoimmune encephalo-
myelitis in mice (43). How sCD83 is generated is not clear, but
proteolytic shedding of cell surface-associated CD83 or alter-
native splicing has been proposed as a possible mechanism
It is unknown whether intestinal commensal bacteria are
able to induce the release of sCD14 and sCD83 from neonatal
innate immune cells. Moreover, it also remains to be eluci-
dated whether gram-positive and gram-negative bacterial spe-
cies differ in the ability to stimulate the release of these two
proteins. Therefore, we examined whether gram-positive com-
mensal bacteria, including Clostridium perfringens, Staphylococ-
cus aureus, and Lactobacillus rhamnosus, or the gram-negative
commensal bacteria Escherichia coli and Bacteroides fragilis
were able to induce the release of sCD14 or sCD83 from
neonatal blood monocytes or DC. We also analyzed the pos-
sible impact of the virulence factor staphylococcal protein A on
S. aureus-induced production of sCD14 by neonatal innate
immune cells. Moreover, as the role of sCD14 or sCD83 in
regulating human Th2 immune responses to allergens is un-
clear, we investigated whether these two proteins could mod-
ulate birch allergen-induced T-cell differentiation in an in vitro
model of allergic sensitization. Autologous DC and T cells
from cord blood were used, as we have previously shown that
birch allergen extract induces a Th2 profile in this system (4).
MATERIALS AND METHODS
Cell separation. Umbilical cord blood, collected in heparin-containing tubes,
was obtained from unselected, healthy, vaginally delivered babies (atopic status
of the parents unknown) born at the Sahlgrenska University Hospital (Go ¨teborg,
Sweden). The study was approved by the Human Research Ethics Committee of
the Medical Faculty, Go ¨teborg University, Go ¨teborg, Sweden. Mononuclear
cells were isolated by density gradient centrifugation over Ficoll-Hypaque (Phar-
macia Biotech, Uppsala, Sweden). Monocytes were isolated by a positive selec-
tion technique, according to the manufacturer’s instructions (MACS system;
Miltenyi Biotech, Bergisch Gladbach, Germany). For isolation of blood DC, a
DC isolation kit (BDCA-1) was used (Miltenyi Biotech) and T cells were purified
with the Dynabead CD4 Positive Isolation kit (Dynal Biotech ASA, Oslo, Nor-
way), according to the manufacturer’s instructions. Isolated T cells were gradu-
ally cooled to ?70°C. After 24 h, the T cells were transferred to a ?143°C
Bacterial strains. Bacterial strains were obtained from the commensal intes-
tinal flora of healthy Swedish infants. Fecal samples were cultured quantitatively
for all major groups of aerobic and anaerobic bacteria and speciated as previ-
ously described (2). In brief, staphylococci were isolated from Staphylococcus
agar and divided into coagulase-positive (S. aureus) and coagulase-negative
staphylococci by the coagulase test. Enterobacteria were isolated on Drigalski
agar and speciated with the API20E biotyping system (API Systems SA, La
Balme les Grottes, France). All isolates obtained from anaerobic cultures were
first checked for the inability to grow under aerobic conditions. Bacteroides spp.
were isolated from Bacteroides bile esculin agar and speciated by Rapid ID 32A
(API Systems). Straight gram-positive rods isolated on Rogosa agar were defined
as lactobacilli, which was confirmed by PCR using group- and species-specific
primers (3). Clostridia, defined as straight gram-positive or gram-labile rods with
or without spores, were speciated with Rapid ID32A. We also used S. aureus
strain Newman and a previously described mutant (DU5873; staphylococcal
protein A-deficient (SpA), derived from Newman), kindly provided by T. Foster,
Department of Microbiology, Trinity College, Dublin, Ireland (32). All bacterial
strains used were washed in phosphate-buffered saline (PBS) (1,000 ? g, 10 min),
counted in a microscope, inactivated by exposure to UV light for 20 min (inac-
tivation was confirmed by a negative viable count), and finally stored at ?70°C.
Bacterial stimulation of cord blood monocytes and cord blood DC. Monocytes
or DC (1 ? 106/ml) were stimulated with UV-killed commensal bacteria, includ-
ing Clostridium perfringens, Staphylococcus aureus, Lactobacillus rhamnosus,
Escherichia coli, or Bacteroides fragilis (1 ? 107bacteria/ml), under serum-free
conditions for 24 or 48 h at 37°C in 5% CO2. Monocytes or DC (1 ? 106/ml) were
also stimulated with S. aureus strain Newman or the SpA mutant strain DU5873
(1 ? 106/ml) under serum-free conditions for 48 h. Phenotypic analysis of DC
stimulated with bacteria was performed by flow cytometry. The cells were sus-
pended in PBS containing 1% fetal calf serum, 0.1% sodium azide, and 0.5 mM
EDTA (fluorescence-activated cell sorter [FACS] buffer), placed in 96-well V-
bottom plates, and pelleted by centrifugation (3 min at 300 ? g, 4°). All of the
monoclonal antibodies (MAbs) used were diluted in FACS buffer at optimal
concentrations. The following MAbs were used: APC–anti-CD11c (B-ly6), PE–
anti-CD14 (M5E2), or PE–anti-CD83 (HB15e) (BD-Bioscience, Stockholm,
Sweden). As isotype controls, mouse monoclonal IgG1 antibodies were used (BD
Bioscience). The cells were incubated with the respective MAbs for a minimum
of 15 min at 4°C in the dark, followed by two washing steps and a final resus-
pension step in FACS buffer before analysis. We analyzed approximately 1,000 to
5,000 cells in a FACSCalibur (BD Bioscience) equipped with CellQuest software
(BD Bioscience). Phenotypic analysis of stimulated monocytes was not per-
formed, as this cell population adheres strongly to plastic during culture. The
release of sCD14 by monocytes and DC was higher after 48 h of stimulation than
after 24 h (see Fig. S1A and B in the supplemental material). Soluble CD83 was
released only by DC, and not by monocytes, and could be measured only after
48 h of stimulation (see Fig. S2A and B in the supplemental material). The
expression of CD14 on the cell surface was higher after 48 h of stimulation than
after 24 h (see Fig. S3A in the supplemental material). The cell surface expres-
sion of CD83, on the other hand, was higher after 24 h of stimulation than after
48 h (see Fig. S3B in the supplemental material).
Reagents. Birch allergen (Betula verrucosa) extract was provided by ALK-
Abello ´ (Hørsholm, Denmark). The proportion of the protein in the allergen
extract was 67.3%, and our batch of allergen extract added 5 pg of LPS per 200
?l of culture medium. Recombinant human CD14 was purchased from R&D
Systems (Minneapolis, MN) and added 2 or 20 pg of LPS per 200 ?l of culture
medium. Recombinant human sCD83 added less than 115 pg of LPS per 200 ?l
of culture medium. Human serum albumin (HSA) was obtained from Octa-
pharma (Stockholm, Sweden). The endotoxin content was assessed by a chro-
mogenic Limulus amoebocyte lysate endpoint test (Chomogenix AB, Mo ¨lndal,
Autologous DC–T-cell cocultures. For generation of immature monocyte-de-
rived DC (MD-DC), monocytes (106cells/ml) were cultured in RPMI 1640
medium (BioWhittaker, Cambrex Company, Belgium) supplemented with 2%
autologous serum, 1 mM L-glutamine, 50 ?g/ml gentamicin, 500 U/ml recombi-
nant IL-4 (R&D Systems), and 800 U/ml recombinant granulocyte-macrophage
colony-stimulating factor (GM-CSF) (R&D Systems). The cells were cultured
for 6 to 7 days and were refed with IL-4 and GM-CSF containing medium every
second day. MD-DC used for DC–T-cell cocultures were either frozen or stim-
ulated with birch allergen alone (50 ?g/ml) or birch allergen in combination with
sCD14 (0.1 or 1 ?g/ml), sCD83 (10 ng/ml), or HSA (0.1 ?g/ml, 1 ?g/ml, or 10
ng/ml) in the presence of tumor necrosis factor (TNF) (20 ng/ml) (R&D Sys-
tems), IL-1? (10 ng/ml) (R&D Systems), and prostaglandin E2(1 ?g/ml) (Sigma-
Aldrich, St. Louis, MO) under serum-free conditions. After 24 h of stimulation,
MD-DC were washed and cocultured (10 ? 103to 15 ? 103cells/well) with
autologous naı ¨ve CD4?T cells (1.5 ? 105cells/well) in serum-free X-Vivo15
medium (BioWhittaker). After 3 days of culture, 100 ?l of the supernatant was
replaced with fresh medium containing IL-2 (50 U/ml) (Nordic BioSite, Ta ¨by,
Sweden). On day 6, frozen autologous MD-DC were thawed and stimulated as
described above and thereafter used on day 7 for restimulation of the T cells.
After 10 days of culture, supernatants were collected for analysis of secreted
cytokines by enzyme-linked immunosorbent assay (ELISA). For analysis of in-
tracellular expression of cytokines, T cells were stimulated with phorbol myris-
tate acetate (10 ng/ml) and ionomycin (1 ?g/ml) for 4 h, and GolgiPlug was
added for the last 3 h. Cells were fixed with paraformaldehyde (2%) and per-
meabilized with saponin (0.5%), and intracellular cytokines were detected by
flow cytometry using fluorescein isothiocyanate–anti-IFN-? (B27) and PE–anti-
IL-13 (JES10-5A2) MAbs (Becton-Dickinson, Erembodegum, Belgium). All re-
agents were purchased from Sigma-Aldrich except for GolgiPlug, which was
obtained from Pharmingen.
ELISAs. Concentrations of IL-5, IL-13, IFN-?, or sCD14 in cell culture su-
pernatants were determined by a standard ELISA procedure, as described in
detail elsewhere (23). Costar plates (Invitrogen, San Diego, CA) were coated
with the following capture MAbs: anti-IL-5 (TRFK5), anti-IL-13 (JES10-5A2),
anti-IFN-? (NIB42), or anti-sCD14 (55-3). Standard curves were generated with
recombinant human IL-5, IL-13, IFN-?, or CD14, respectively. All antibodies
and standards were purchased from Pharmingen except for recombinant CD14,
which was obtained from R&D Systems. The following biotinylated detection
antibodies were used: anti-IL-5 (JES1-5A10), anti-IL-13 (B69-2), anti-IFN-?
4098 LUNDELL ET AL.INFECT. IMMUN.
(4S.B3), or anti-sCD14 (3-C39). Samples, standards, biotinylated antibodies, and
streptavidin-horseradish peroxidase were diluted in the high-performance
ELISA buffer Sanquin (Amsterdam, The Netherlands). In control experiments,
we found that preincubation of sCD14 with various doses of LPS from E. coli
(Sigma-Aldrich) or peptidoglycan from S. aureus (Sigma-Aldrich) did not inter-
fere with the detection of sCD14 in the ELISAs (see Fig. S4 in the supplemental
Concentrations of sCD83 were determined with a modification of a previously
described ELISA (18, 19). Costar plates (Invitrogen, San Diego, CA) were
coated with monoclonal anti-CD83 (clone HB15a;Immunotech, Marseille,
France). The isotype-matched CD69 control MAb (Immunotech) was used to
provide a measure of the nonspecific background for each individual sample. The
capture antibodies CD83 and CD69 were diluted in PBS, and the plates were
thereafter blocked with 10% goat serum (Gibco-BRL, Life Technologies, New
Zealand). Standard curves were generated with recombinant sCD83 (CD83-
GST) (29). For detection, polyclonal rabbit anti-CD83 (RA83; kindly provided
by B. Hock, Christchurch Hospital, New Zealand) was diluted in 5% goat serum,
2% mouse serum, and 1% dried nonfat milk in PBS to a concentration of 10
?g/ml (18, 19, 29). Thereafter, biotinylated monoclonal mouse anti-rabbit anti-
bodies (RG-96;Sigma-Aldrich), diluted in reagent buffer, were added to the
plates. Next, the plates were incubated with streptavidin-horseradish peroxidase
(Sanquin, The Netherlands) diluted in PBS containing 0.5% bovine serum albu-
min. Then, 3,3?5,5?-tetramethylbenzidine (Dako, Carpinteria, CA) substrate was
added to the plates, which were kept in the dark, and the reaction was stopped
by the addition of 2.5 M H2SO4.
Statistical analysis. The data were analyzed by the Kruskal-Wallis test or the
Friedman test, followed by Dunn’s multiple-comparison test or the Wilcoxon
matched-pairs test, as described in the figure legends (GraphPad Prism, San
Production of sCD14 and expression of CD14 by neonatal
innate immune cells in response to commensal bacteria. As
the physiological stimulus that may trigger the production of
sCD14 and sCD83 is unclear, we investigated whether human
neonatal innate immune cells would release sCD14 or sCD83
in response to stimulation with commensal intestinal bacteria
isolated from Swedish infants. Cord blood monocytes or DC
were exposed to the UV-killed commensal gram-positive bac-
terial strain C. perfringens, S. aureus, or L. rhamnosus or the
gram-negative bacterial strain E. coli or B. fragilis.
We found that cord blood monocytes released significantly
larger amounts of sCD14 after stimulation with the gram-
positive C. perfringens or S. aureus but not with the gram-
negative bacteria, compared to unstimulated cells (Fig. 1A).
Freshly isolated cord blood DC, on the other hand, released
sCD14 in response to both the gram-positive C. perfringens or
S. aureus and to the gram-negative E. coli or B. fragilis, as
shown in Fig. 1B. In Fig. 1C, we show that CD14 was upregu-
lated on the cell surface of blood DC when exposed to both
gram-negative and gram-positive bacteria, but CD14 was not
detected intracellularly either in cells exposed to bacteria or in
unstimulated cells (see Fig. S5 in the supplemental material).
FIG. 1. Release of sCD14 and expression of CD14 on the cell surface in response to bacterial stimulation. Shown is the release of sCD14 by
cord blood monocytes (A) or cord blood DC (B) in response to the UV-killed gram-positive bacterium C. perfringens, S. aureus, or L. rhamnosus
or the gram-negative bacterium E. coli or B. fragilis for 48 h. (C) Expression of CD14 on the cell surface by DC after exposure to the bacteria listed
above for 48 h. Each symbol represents one individual (panel A, n ? 8; panel B, n ? 5 to 10; panel C, n ? 3 to 6), and the horizontal bars represent
the median. (D) Dot plots represent CD14 expression on unstimulated cord blood DC and on cells stimulated with S. aureus or E. coli for 48 h.
?, P ? 0.05; ??, P ? 0.01; ???, P ? 0.001 (Kruskal-Wallis followed by Dunn’s multiple comparison test).
VOL. 75, 2007INDUCED sCD14 AND sCD83 INHIBIT Th2 RESPONSES4099
In Fig. 1D, we show representative dot plots regarding CD14
expression on the cell surface of unstimulated cord blood DC
and on cells stimulated with S. aureus or E. coli for 48 h.
To examine the possible impact of staphylococcal protein A
(SpA) on S. aureus-induced sCD14 release from neonatal
monocytes and DC, the cells were stimulated with a wild-type
S. aureus strain or an SpA-deficient S. aureus strain (isogenic
strains with respect to SpA). In Fig. 2, we show that the SpA-
deficient S. aureus strain triggered significantly lower levels of
sCD14 from both cord blood monocytes and DC than did the
wild-type strain. Taken together, freshly isolated cord blood
monocytes appear to release sCD14 only when exposed to
gram-positive bacteria, whereas the sCD14 release from DC
and the CD14 expression on the DC cell surface was upregu-
lated in response to both gram-positive and gram-negative
bacteria. Furthermore, SpA seems to be one factor that influ-
ences sCD14 production by neonatal innate immune cells after
S. aureus stimulation.
Production of sCD83 and expression of CD83 by neonatal
cells in response to bacteria. When we examined the ability of
innate immune cells to release sCD83 in response to commen-
sal bacteria, we found that the blood DC released sCD83 in
response to all gram-positive and gram-negative bacterial spe-
cies we included in the experiments (Fig. 3A), whereas CD83
expression on the cell surface seemed to be upregulated only in
response to stimulation with the gram-positive C. perfringens,
S. aureus, or L. rhamnosus (Fig. 3B). In Fig. 3C, we show
representative dot plots regarding CD83 expression on the cell
surface of unstimulated cord blood DC and on cells stimulated
with S. aureus or E. coli for 48 h. CD83 was detected intracel-
lularly in the majority of the blood DC, but there were no
differences between stimulated and unstimulated cells (Fig.
3D). In contrast to sCD14, monocytes did not release sCD83
when exposed to bacteria (see Fig. S2B in the supplemental
material). In summary, cord blood DC released sCD83 when
exposed to both gram-positive and gram-negative bacteria,
whereas the cell surface expression of CD83 was upregulated
only in response to the gram-positive bacteria.
Soluble CD14 and sCD83 suppress human neonatal birch
allergen-induced IL-13. To investigate whether sCD14 or
sCD83 might modulate neonatal birch allergen-induced T-cell
responses, DC were stimulated with birch allergen extract
alone or in the presence of sCD14, sCD83, or the control
protein HSA and were thereafter cocultured with autologous
CD4?T cells. DC were stimulated in the presence of TNF,
IL-1?, and prostaglandin E2, as we have previously shown that
DC need to be fully mature to be able to induce birch allergen-
induced T-cell expansion and differentiation (4). In order to
use physiological doses of sCD14 in our culture systems, the
selected concentrations (0.1 or 1 ?g/ml) were based on the
median levels that we have recently found to be present in
the circulation during infancy (cord, 0.18 ?g/ml; 4 months, 0.57
?g/ml; 18 months, 0.97 ?g/ml; 3 years, 0.55 ?g/ml) (30). Re-
garding sCD83, we used a higher concentration (10 ng/ml)
than the median levels that were present in the circulation
during infancy (cord, 0.1 ng/ml; 4 months, 0.14 ng/ml; 18
months, 0.1 ng/ml; 3 years, 0.1 ng/ml) (30), as it has been shown
that sCD83 is elevated at the local site of inflammation but not
in the circulation (20).
In Fig. 4A, we show that birch alone induced significantly
higher levels of the Th2 cytokine IL-13 than did unstimulated
cells. Birch in combination with the higher dose of sCD14 (1
?g/ml) or sCD83 (10 ng/ml) induced significantly lower levels
of IL-13 relative to birch alone, whereas the levels of IL-13
induced by birch together with the lower dose of sCD14 (0.1
?g/ml) did not differ significantly from those induced by birch
allergen alone. Soluble CD14 or CD83 alone did not affect the
production of IL-13 relative to unstimulated cells. Neither
sCD14 nor sCD83 appeared to affect the viability of the cells as
judged by staining with trypan blue. Birch in combination with
the control protein HSA did not affect IL-13 production com-
pared to birch alone (Fig. 4B). The production of IL-5 and
IFN-? was also measured in the cell cultures, but only very low
levels of IL-5 and no IFN-? were induced, and increased levels
were not seen after stimulation (see Fig. S6A and B in the
supplemental material), as previously shown with neonatal T
cells (4, 27).
To support our observations regarding the secretion of IL-13
into the cell culture supernatant, we also examined the intra-
cellular IL-13 expression in CD3-positive T cells in response to
DC stimulated with birch alone or birch in combination with
sCD14 or sCD83 in a limited number of experiments (see
Table S1 in the supplemental material). In Fig. 4C, we show
data from one experiment after stimulation with birch relative
to unstimulated cells and the suppressive effect of adding 1
?g/ml of sCD14 to the cultures. In Fig. 4D, we show the
corresponding data for an experiment using sCD83. Taken
together, stimulation of DC with birch in combination with
sCD14 or sCD83 resulted in suppression of IL-13 production,
manifested by reduced levels in the supernatant, a finding
which was supported by lower numbers of T cells harboring
IL-13 intracellularly in the majority of the experiments.
Many epidemiological studies indicate that exposure to high
levels of microbes early in life may induce tolerance to envi-
ronmental allergens and thereby prevent allergy development
FIG. 2. S. aureus-induced release of sCD14 is influenced by staph-
ylococcal protein A. Shown are levels of sCD14 released by cord blood
monocytes (A) or DC (B) in response to a wild-type S. aureus strain or
a staphylococcal protein A mutant after stimulation for 48 h. Each
symbol represents one individual (panel A, n ? 6; panel B, n ? 8), and
the horizontal bars represent the median. ?, P ? 0.05 (Wilcoxon
4100LUNDELL ET AL.INFECT. IMMUN.
(11, 12). The immunological mechanisms behind this phenom-
enon are not clear, but stimulation of the developing immune
system may be an important factor. Here we demonstrate that
gram-positive, but not gram-negative, commensal intestinal
bacteria triggered cord blood monocytes to release sCD14.
Neonatal DC, on the other hand, released sCD14 and sCD83
when exposed to both gram-positive and gram-negative bacte-
ria. Moreover, both sCD14 and sCD83 suppressed human neo-
natal birch allergen-induced Th2 differentiation in an autolo-
gous in vitro model for allergen sensitization. Thus, we show
for the first time how the production of sCD14 and sCD83 is
differentially stimulated by gut bacteria and that both proteins
modulate neonatal allergen-induced T-cell responses.
We have recently found that children with food allergies are
less frequently colonized with S. aureus in the gut at 2 weeks of
age, and they also tend to have lower levels of sCD14 in plasma
than do healthy children (30). No other groups have studied
the effects of early intestinal bacterial colonization in relation
to the development of food allergies. However, one study has
shown that S. aureus is more frequent in the gut of 6-month-old
children who develop atopic dermatitis than in children who
remain healthy (9). Interestingly, in this study no difference in
S. aureus colonization between the groups was seen at 1 week
or 1 month of age. This is supported by data from our study
showing no relation between early S. aureus colonization and
the development of eczema, in contrast to the development of
food allergies (30). One possible explanation for the increased
frequency of intestinal S. aureus colonization at 6 months of
age could be that the children had at that time developed S.
aureus-infected atopic dermatitis, which could in turn lead to
increased S. aureus colonization of the gut.
In our previous study, we also demonstrated that children
who are colonized with S. aureus early in life have higher levels
of sCD14 in the circulation than do noncolonized children,
whereas early colonization with E. coli had no effect (30).
However, a cause-effect relationship between S. aureus coloni-
zation and increased sCD14 levels in the circulation has not
been proven in vivo. Here we demonstrate that neonatal
monocytes and DC released sCD14 in response to the gram-
positive bacteria C. perfringens and S. aureus. In contrast to
monocytes, cord blood DC released sCD14 in response to both
gram-positive and gram-negative gut bacteria. Thus, C. perfrin-
gens and S. aureus, but not E. coli or B. fragilis, seem to have
characteristics that trigger both monocytes and DC to release
FIG. 3. Release of sCD83 and expression of CD83 on the cell surface in response to bacterial stimulation. (A and B) Release of sCD83 and
expression of CD83 on the cell surface by blood DC in response to the UV-killed gram-positive bacterium C. perfringens, S. aureus, or L. rhamnosus
or the gram-negative bacterium E. coli or B. fragilis for 48 h. Each symbol represents one individual (panel A, n ? 5 or 6; panel B, n ? 3 to 6),
and the horizontal bars represent the median. (C) Dot plots represent CD83 expression on unstimulated cord blood DC and on cells stimulated
with S. aureus or E. coli for 48 h. (D) Intracellular expression of CD83 by blood DC in response to the UV-killed gram-positive bacterium C.
perfringens, S. aureus, or L. rhamnosus or the gram-negative bacterium E. coli or B. fragilis for 48 h. Each symbol represents one individual (n ?
3), and the horizontal bars represent the median. ?, P ? 0.05; ??, P ? 0.01 (Kruskal-Wallis followed by Dunn’s multiple comparison test).
VOL. 75, 2007INDUCED sCD14 AND sCD83 INHIBIT Th2 RESPONSES 4101
sCD14. This might be explained by the fact that monocytes and
DC express different repertoires of pattern recognition recep-
tors (22, 24, 38). Monocytes express CD14 and TLR2 with high
intensity and TLR4 with intermediate intensity on the cell
surface relative to DC, which express very low levels of these
three receptors (24). Moreover, it has been shown that TLR2
recognizes structures expressed only by gram-positive bacteria,
such as lipoteichoic acid (37), which is in accordance with our
results demonstrating that monocytes release sCD14 only in
response to gram-positive bacterial stimulation. Furthermore,
our group has previously shown that the pattern of proinflam-
matory cytokine responses to gut bacteria is changed when
monocytes differentiate to DC (24). Monocytes produced
higher levels of IL-12p70 and TNF after stimulation with the
gram-positive bacterium L. plantarum relative the levels in-
duced by the gram-negative bacterium E. coli, whereas DC
secreted large amounts of IL-12p70 and TNF in response to E.
coli (24). In line with these results, it has been shown that
mucosal explants from the human gut produce TNF in re-
sponse to stimulation with E. coli but not when exposed to
lactobacilli (10). Taken together, not only cytokine profiles but
also production of other immunoregulatory proteins, such as
sCD14, are changed when monocytes differentiate to DC.
The ability of the gram-positive bacteria C. perfringens and S.
aureus to induce high levels of sCD14 might be related to their
ability to elicit TNF production. Our group and others have
shown that gram-positive bacteria induce higher levels of TNF
from cord blood mononuclear cells, peripheral blood mono-
nuclear cells, and purified monocytes than do gram-negative
bacteria (17, 23, 24), whereas there were no differences in IL-6
production (23). Indeed, the most potent gram-positive bacte-
rium to induce TNF production by monocytes was found to be
S. aureus (17). In the present study, we demonstrate that the S.
aureus-induced release of sCD14 from neonatal blood mono-
cytes and DC was influenced by the expression of SpA on the
bacterial cell surface. One possible explanation for this finding
could be that SpA has been shown to bind to the TNF receptor
and may thereby directly trigger the release of sCD14 (16).
However, it remains to be elucidated why C. perfringens, which
is a poor inducer of TNF, was found to be the most potent
inducer of sCD14 by neonatal monocytes and DC (17).
We demonstrate that gram-positive and gram-negative com-
FIG. 4. Soluble CD14 and sCD83 suppress birch allergen-induced IL-13. DC were stimulated with birch, birch plus sCD14, birch plus sCD83,
or birch plus HSA and cocultured with autologous CD4?T cells. (A) On day 10, production of IL-13 was analyzed by ELISA, and boxes and
whiskers summarize data from 11 subjects. The boxes show the median and the interquartile range, and the whiskers show the range. (B) IL-13
production induced by birch allergen compared to birch plus HSA (n ? 7). (C) Intracellular expression of IL-13 in T cells after 10 days of coculture
with DC stimulated with birch, birch plus sCD14, birch plus HSA, or sCD14 alone. Data demonstrate one experiment out of four. (D) Intracellular
expression of IL-13 in T cells after 10 days of coculture with DC stimulated with birch, birch plus sCD83, birch plus HSA, or sCD83 alone. Data
demonstrate one experiment out of two. The intracellular IL-13 expression was analyzed by flow cytometry, and the percentages of positive cells
are indicated in the upper right quadrant. ?, P ? 0.05; ??, P ? 0.01 (Friedman test followed by Dunn’s multiple comparison test).
4102 LUNDELL ET AL.INFECT. IMMUN.
mensal gut bacteria induce release of sCD83 from cord blood
DC but not from monocytes. Our results are consistent with a
previous in vitro study showing that human monocyte-derived
DC from adult individuals release sCD83 when stimulated with
LPS (19). Previously, we found that sCD83 is present in small
amounts in the circulation of human infants compared to the
levels of sCD14 (30). Interestingly, clinical studies have re-
ported that the levels of soluble CD14 are elevated in both
plasma and synovial fluid from patients with rheumatoid ar-
thritis (8, 40), but the levels of sCD83 are increased only in the
synovial fluid, not in the circulation (20). Thus, the amount of
sCD14 appears to be increased both locally and systemically
during immune activation, whereas elevated levels of sCD83
may be found only at the local site of inflammation. This may
explain why we found no relation between the levels of sCD83
in the circulation and early intestinal colonization in spite of
the fact that the bacteria were able to induce release of sCD83
from DC in vitro (30). Therefore, sCD83 may well be elevated
locally in the intestinal mucosa in response to the commensal
Soluble CD14 appears to have several immunological func-
tions. In addition to its role as a receptor for bacterial struc-
tures, including LPS and peptidoglycan, sCD14 modulates cel-
lular immune responses by suppressing T-cell proliferation and
production of IFN-? and IL-4 (34). Accordingly, we found that
sCD14 suppressed birch allergen-induced IL-13 production
and decreased the number of IL-13 expressing T cells. One
explanation for the observed regulatory function of sCD14 on
T-cell activation could be via an inhibitory effect on IL-2 pro-
duction (34). As for sCD14, we found that sCD83 inhibited
birch allergen-induced Th2 differentiation in our in vitro
model for allergic sensitization. Our observation is in line with
those of others, as it was recently shown that sCD83 has im-
munoregulatory functions by inhibiting disease symptoms in a
murine model of experimental autoimmune encephalomyelitis
in vivo, which is dominated by a Th1-type response (43). Fur-
ther, sCD83 inhibits human DC-mediated allogeneic T-cell
proliferation in vitro in a dose-dependent manner (29). One
possible explanation for the effect of sCD83 on T-cell differ-
entiation could be by affecting the activation of DC. Indeed,
sCD83 has been found to influence the arrangement of the
cytoskeleton of DC and thereby inhibiting DC–T-cell cluster-
ing, a prerequisite for DC-mediated T-cell expansion (25).
Thus, our results, in combination with those of others, show
that sCD14 and sCD83 are able to suppress both Th2 and Th1
responses, suggesting that these soluble proteins may have an
important function in maintaining immune homeostasis.
In summary, we show that neonatal monocytes release
sCD14 and neonatal DC release sCD14 and sCD83 when stim-
ulated with commensal gut bacteria. Moreover, both proteins
suppressed birch allergen-induced Th2 differentiation in an in
vitro model for allergen sensitization by inhibiting the produc-
tion and expression of IL-13, an important switch factor for
IgE. Whether the present study may help to explain the im-
munological mechanisms at the cellular level behind recent
epidemiological studies needs to be further elucidated. How-
ever, we suggest that sCD14 and sCD83 may be mediators that
are induced by the intestinal microflora and that may be in-
volved in down-regulating immune responses, leading to aller-
gic diseases in children.
This work was supported by the Swedish Research Council, by the
Vårdal Foundation, by Cancer-och Allergifonden, by the Swedish
Asthma and Allergy Association Research Foundation, by Medi-SAM
(ALF), by Frimurare Barnhusdirektionen, by Åke Wibergs Stiftelse, by
Konsul Th C Berghs Stiftelse, and by Stiftelsen Tornspiran. A.S. was
supported by the Deutsche Forschungsgemeinschaft, Sonderforschun-
gsbereich SFB 643, grant A4.
We are deeply grateful to the staff at the Delivery Units of Sahlg-
renska University Mo ¨lndal Hospital for collecting cord blood samples.
We also thank Ulla Seppa ¨la ¨, of ALK-Abello ´, for assistance with the
birch allergen extract and Erika Lindberg, of the Department of Clin-
ical Bacteriology of Go ¨teborg University, for technical assistance with
the bacterial strains.
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Editor: J. F. Urban, Jr.
4104LUNDELL ET AL.INFECT. IMMUN.