Second Division, Department of Internal Medicine, Hamamatsu University School of Medicine, Hamamatsu, Shizuoka, Japan
Correspondence: Kingo Chida, M.D., Ph.D., 3600 Handa-cho, Hamamatsu, Shizuoka 431-3192 Japan. E-mail: chidak11{at}hama-med.ac.jp
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, IL-6, IL-8, IL-10, and IL-12 (p40) by human DCs in
a dose-dependent manner. Furthermore, MDP-Lys-treated DCs showed
enhanced antigen-presenting function compared with untreated DCs, as
assessed by an allogeneic mixed lymphocyte reaction. These results
suggested that the immunoadjuvant activity of MDP-Lys in vivo is
mediated, in part, by its stimulation of DC function.
Key Words: immunoadjuvant activity • antigen-presenting cells • T cell immunity
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DCs have a highly developed function in the immune system as specialized APCs for the primary immune response [23 , 24 ]. DCs act as sentinels and are widely distributed in virtually all organs [25 ]. They are strategically positioned to take up antigens, after which they migrate to lymphoid organs, where they present the antigens to naive T cells, leading to the initiation of T cell immunity [23 ]. The ability of DCs to act as potent APCs is largely attributable to their strong expression of MHC and costimulatory molecules as well as their capacity to produce various cytokines. Recently, factors present during the innate phase of the immune response, such as bacterial products and proinflammatory cytokines, have been reported to enhance the expression of MHC and costimulatory molecules and to induce cytokine production by DCs, leading to their activation [26 27 28 29 ]. Therefore, we considered it possible that MDP-Lys could directly augment DC function and that this activity might also be implicated in its immunostimulatory effect in vivo.
Thus, this study was conducted to explore the effects of MDP-Lys on DC
function. For this purpose, using human DCs generated by culturing
peripheral blood cells in the presence of IL-4 and
granulocyte-macrophage (GM)-CSF, we examined their expression of
surface molecules, production of cytokines, and allostimulatory
capacity after treatment with MDP-Lys. We found that MDP-Lys markedly
up-regulated the expression of CD80, CD83, CD86, and CD40 and
stimulated the production of TNF-, IL-6, IL-8, IL-10, and IL-12
(p40) by human DCs, resulting in enhancement of their
antigen-presenting function. These results suggest that the activation
of DCs by MDP-Lys is involved in its immunoadjuvant activities in vivo.
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Cell preparations
Peripheral blood mononuclear cells (PBMCs) were isolated from
heparinized whole blood of normal healthy donors by density gradient
centrifugation with a Lymphoprep centrifuge (Nycomed, Oslo, Norway).
PBMCs were harvested from the interface and washed twice in
phosphate-buffered saline (PBS) supplemented with 5 mM EDTA and 0.5%
bovine serum albumin (Sigma). Subsequently, CD14+ cells
were separated by magnetic sorting with a MACS cell sorter (Miltenyi
Biotec, Berglsh Gladbach, Germany) according to the manufacturer’s
instructions. Briefly, PBMCs were incubated with saturating
concentrations of anti-CD3, anti-CD19, and anti-CD56 monoclonal
antibodies (mAbs) conjugated with superparamagnetic microbeads for 15
min on ice and then washed in PBS containing 5 mM EDTA and 0.5% human
serum. Unlabeled cells were then isolated by elution from magnetic
columns, routinely resulting in >98% purity of CD14+
cells as assessed by flow cytometric analysis.
Generation of DCs from CD3- CD19-
CD56- cell culture
Isolated CD3- CD19- CD56-
cells (2x106/mL) were cultured in 24-well tissue culture
plates (Costar, Cambridge, MA) in 1 mL of the culture medium containing
1,000 U/mL of GM-CSF, 1,000 U/mL of IL-4, and 1% human plasma
(complete medium) at 37°C in a 5% CO2 incubator. Every 2
days, half the medium was removed and an equivalent volume of fresh
complete medium was added. For most experiments, the cells were
collected routinely after 7–10 days of culture. The cells thus
obtained contained >95% DCs as assessed by morphology analysis and
flow cytometric analysis with anti-CD1a mAb. For stimulation with
MDP-Lys, after 7 days of culture, nonadherent cells were harvested,
washed, and subcultured in concentrations of 106 cells/mL
in 24-well plates in 1 mL of complete medium with or without various
doses of MDP-Lys for 72 h.
Flow-cytometric analysis
For immunophenotyping, cultured cells were analyzed by
dual-color flow cytometry. The cells were washed in PBS and incubated
with the appropriately diluted phycoerythrin (PE)-conjugated anti-CD1a
[clone BL6, mouse immunoglobulin (Ig)G1] (Immunotech, Marseilles,
France), anti-CD83 (clone HB15a, mouse IgG2b) (Immunotech), anti-CD40
(clone MAB89, mouse IgG1) (Immunotech), anti-CD80 (clone MAB104, mouse
IgG1) (Immunotech), anti-CD86 (clone IT2.2, mouse IgG2b) (PharMingen,
San Diego, CA), and fluorescein isothiocyanate (FITC)-conjugated
anti-human leukocyte antigen-DR (HLA-DR) (clone Immu-357, mouse IgG1)
(Immunotech) mAbs for 30 min on ice. Parallel incubations were also
performed with FITC- or PE-conjugated irrelevant antibodies matched for
the isotypes as controls. The cells were washed in PBS and then
analyzed with an EPICS® Profile-II flow cytometer (Beckman Coulter,
Fullerton, CA). The expression of cell surface markers was evaluated in
terms of the percentage of positive cells and the mean fluorescence
intensity (MFI). The cutoff level for the definition of positive cells
was thus set so that <1% of irrelevant antibody-stained cells were
positive.
Cytokine assays
The levels of the cytokines IL-6, IL-8, IL-10, IL-12 (p40), and
TNF- in the culture supernatants were measured using enzyme-linked
immunosorbent assay kits from R&D Systems (Minneapolis, MN).
Allogeneic MLR
Stimulator cells from DC cultures that had been cultured for
72 h with MDP-Lys or LPS were harvested, washed, and irradiated
(2,000 rad). Allogeneic T cells were prepared from PBMCs using a T cell
Recovery Column (Hornby, Canada). Subsequently, CD45RA+
cells were purified from the allogeneic T cell populations by magnetic
cell sorting with anti-CD45RA mAbs conjugated with super-paramagnetic
microbeads (Miltenyi Biotec). Various numbers of DCs were then placed
in 96-well flat-bottom tissue culture plates (Corning, Acton, MA) alone
or with allogeneic CD45RA+ T cells (2x105
cells/well). The allogeneic mixed leukocyte reaction (MLR) was carried
out in RPMI 1640 supplemented with 10% heat-inactivated normal human
AB serum, 10 µg/mL of gentamycin, 25 mM HEPES, and 50 µM
2-mercaptoethanol (Sigma) and incubated at 37°C in an atmosphere with
5% CO2. On day 5, the cells were pulsed with
[3H]thymidine (1 µCi/well) (Amersham Japan, Tokyo) for
16 h. The cultures were then harvested with a cell harvester, and
the incorporated radioactivity was counted in a liquid scintillation
counter (LSC-3100; Aloka Co. Ltd., Japan).
Statistics
For the statistical analysis, the Mann-Whitney test was used. A
P value <0.05 was considered significant. All data are
expressed as mean ± SE unless otherwise specified.
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 View larger version (36K): [in a new window] |
Figure 1. Flow cytometric analysis of cultured human DCs. CD3-
CD19- CD56- blood cells were cultured for 7
days with IL-4 (1,000 U/mL) and GM-CSF (1,000 U/mL). Cells were labeled
with HLA-DR(FITC) and CD1a(PE), CD83(PE), CD80(PE), CD86(PE), or
CD40(PE) mAbs. Data from one representative experiment out of 14 are
shown.
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1.0 ng/mL) was greater than that stimulated by MDP (1,000 ng/mL)
(Fig. 3) .
 View larger version (27K): [in a new window] |
Figure 2. Effects of MDP-Lys on the profiles of surface molecules expressed by
human DCs. DCs incubated for 72 h in medium alone (line 2) or
medium containing 1,000 ng/mL of MDP-Lys (line 3) were analyzed by flow
cytometry for expression of HLA-DR, CD1a, CD83, CD80, CD86, and CD40.
Line 1 indicates isotype controls. One representative experiment out of
14 is shown.
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 View larger version (21K): [in a new window] |
Figure 3. Effects of MDP-Lys on the phenotype of human DCs. DCs were cultured for
72 h in medium alone, medium containing various concentrations of
MDP-Lys, or LPS. DCs were then stained with the indicated mAbs, and
their surface molecule expression was analyzed by flow cytometry.
Results are expressed as MFI. Data are expressed as the mean ±
SE of >10 independent experiments. *, P <
0.05; **, P < 0.01; ***, P < 0.001
compared with MFI of untreated DCs.
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, IL-6, IL-8, IL-10, and IL-12
(p40) in culture supernatants after stimulation with MDP-Lys. The
production of cytokines was examined after 72 h of treatment with
MDP-Lys because cytokine levels reached a plateau at this time (data
not shown). We found that even unstimulated DCs produced low levels of
these cytokines (Table 1
). In response to the addition of various concentrations of
MDP-Lys, the production of TNF-, IL-6, IL-8, and IL-12 (p40) was
significantly increased in a dose-dependent manner, with a significant
effect already observed at a concentration of 1 ng/mL (Table 1)
.
MDP-Lys also tended to induce a dose-dependant release of IL-10, but
the effect was not significant (control vs. 1,000 ng/mL;
P=0.098) (Table 1)
. Compared the cytokine producing capacity
of MDP with that of LPS. TNF-, and IL-10 production by
1,000 ng/mL of MDP were approximately comparable to those by
0.1 ng/mL of LPS. Treatment with 1,000 ng/mL of MDP induced the levels
of IL-8 production similar to treatment with 1.0 ng/mL of
LPS. However, the levels of IL-6 and IL-12 (p40) in the
supernatants were much higher in LPS-treated DCs than in MDP-treated
DCs. |
View this table: [in a new window] |
Table 1. Cytokine Production by DCs in Response to MDP-Lys or LPS
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 View larger version (20K): [in a new window] |
Figure 4. MDP-Lys enhances the allostimulatory capacity of human DCs. DCs were
incubated for 72 h in medium alone, 1,000 ng/mL of MDP-Lys, or 0.1
ng/mL of LPS. They were then washed, irradiated (2,000 rad), and added
in various numbers to allogeneic CD45RA+ T cells
(2x105/well) in 96-well flat-bottom microtiter plates.
Thymidine incorporation was measured on day 4 by a 16-h pulse
[3H]thymidine (1 µCi/well). Cultures were set up in
triplicate. Data from one representative experiment out of four are
shown.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Human DCs generated from CD3- CD19-
CD56- blood cells with GM-CSF/IL-4 were shown to exhibit
high levels of expression of HLA-DR, CD1a, CD80, CD86, and potent
antigen-presenting function, as assessed by an allogeneic MLR,
indicating that they had the characteristic features of DCs. Upon
stimulation with MDP-Lys, marked up-regulation of CD80, CD83, CD86, and
CD40 was observed in a dose-dependent manner. Because costimulatory
molecules such as CD80, CD86, and CD40 are particularly important for
optimal activation of primed and unprimed T cells [32
,
33
], DCs stimulated with MDP-Lys are likely to be more
efficient for T cell stimulation. In addition, MDP-Lys might also
augment their expression of CD83, which was recently shown to be a
DC-specific marker expressed only by mature DCs [28
,
31
]. Human DCs generated from peripheral blood with
GM-CSF/IL-4 have been reported to be relatively immature in terms of
their phenotype and antigen-presenting capacity [28
]. In
response to bacterial components (LPS and fixed Staphylococcus
aureus), ligation via the CD40 ligand (CD40L), or inflammatory
cytokines (TNF- and IL-1), these immature DCs were shown to become
fully mature DCs in which there were further increases in the
expression of costimulatory molecules and CD83 [27
,
28
, 34
]. Thus, the finding that MDP-Lys
enhanced the expression of CD80, CD86, CD40, and CD83 on human DCs
suggested that MDP-Lys promoted the phenotypic maturation of these DCs.
It is interesting that we found two populations in MDP-Lys-treated DCs
in terms of CD83 expression, CD83low and
CD83high populations. It is possible that cultured DCs
derived from CD14+ blood cells contain two different
populations with regard to the induction of CD83 expression by MDP-Lys.
In contrast to costimulatory molecules such as CD80, CD86, and CD40,
MDP-Lys did not further increase HLA-DR expression on human DCs,
although the DCs expressed high levels of HLA-DR even without
stimulation. In monocytes and B cells, the data concerning the
regulation of MHC class II expression by MDP have been controversial
[8
, 9
, 35
]. Several studies
failed to demonstrate any MDP-induced enhancement of MHC class II
expression in murine peritoneal macrophages and B cell lines
[8
, 35
]. In contrast, Heinzelmann and
co-workers showed that MDP slightly increased HLA-DR expression on
human monocytes [9
]. Another study analyzed mRNA
induction of HLA-DR by MDP and demonstrated only a minimal increase of
HLA-DR mRNA in human monocytes [36
]. These conflicting
results may be attributable to the type of MDP analogues used, the
nature of the responding cells, or the species employed. Taking our
results together with the previous findings, MDP does not appear to be
a potent inducer of MHC class II antigens.
Recently, it has been shown that DCs can secrete a large array of
cytokines, including IL-6, IL-8, IL-10, IL-12, IL-18, and TNF-, in
response to a variety of stimuli such as bacterial products, phorbol
myristate acetate/ionomycin, and CD40 ligation [27
,
37
]. In addition to the T cell stimulatory property of
DCs via direct cell-to-cell interaction, the cytokine production by DCs
is also thought to play an important role in controlling immunity. In
the present study, we observed that human DCs stimulated with MDP-Lys
could secrete considerable amounts of TNF-; IL-6; IL-8; IL-12 (p40);
and, to a lesser degree, IL-10 in a dose-dependent manner. These
cytokines are known to be crucial for the regulation of inflammatory
and immunologic responses. TNF- is an important proinflammatory
cytokine that up-regulates adhesion molecules and primes T cells
[38
]. IL-6 induces proliferation and differentiation of
B cells as well as stimulation of T cells [39
]. IL-8 is
a potent chemotactic factor for neutrophils and T cells
[40
]. IL-12 is an essential cytokine in promoting the T
helper cell type 1 response [41
]. Thus, it is suggested
that MDP-Lys can induce the release of these cytokines by DCs in vivo,
which, in turn, may augment protective immunity. It is interesting that
MDP-Lys-treated DCs also secreted small amounts of IL-10, which is
known to inhibit T cell proliferation and down-regulate expression of
MHC class II and costimulatory molecules by monocytes and macrophages
[42
, 43
]. IL-10 was also shown to decrease
IL-12 production by DCs and the antigen-presenting function of DCs
[44
]. In agreement with our results, recent studies
using CD14+-derived human DCs showed their capacity to
produce IL-10 in response to LPS and phorbol myristate
acetate/ionomycin [27
, 37
]. Considering the
immunosuppressive role of IL-10, the production of IL-10 by DCs might
be implicated in negatively controlling the level of T cell activation
and DC function itself.
DCs are unique among APCs in their ability to stimulate naive T cells, as measured by an allogeneic MLR. The human DCs used in the present study were shown to induce strongly the proliferation of allogeneic naive T cells without stimulation. However, we found that MDP-Lys could further enhance the capacity of human DCs to stimulate allogeneic naive T cells. The increase in the allostimulatory potential of DCs by MDP-Lys was most noticeable when the cells were cultured using the smallest ratio of stimulators/responders. This suggests that MDP-Lys can augment the antigen-presenting function of DCs and that consequently even a small number of DCs might be able to efficiently provoke T cell-mediated immune responses.
To evaluate the potency of the ability of MDP-Lys to stimulate DC
function in vitro, we compared the stimulatory activity of MDP-Lys with
that of LPS, which is one of the most powerful DC activators
[27
]. In terms of the surface expression of
immunostimulatory molecules and accessory cell function, stimulation
with 0.1 ng/mL of LPS was shown to be comparable to stimulation with
1,000 ng/mL of MDP. It is interesting that LPS increased the expression
of HLA-DR whereas MDP-Lys failed to do so. Although the reason for this
discrepancy is not clear, the difference in signaling mechanism(s)
between MDP-Lys and LPS might be responsible. As to cytokine
production, LPS was shown to have a more potent capacity to produce
cytokines than MDP-Lys. However, MDP-Lys could induce a significant
increase in the production of TNF-, IL-6, IL-8, and IL-12 (p40) in a
dose-dependent manner. Collectively, although the ability of MDP-Lys to
stimulate DCs in vitro was not as powerful as that of LPS, the present
data clearly indicate that MDP-Lys did efficiently activate DC
function. Considering the potential harm of in vivo administration of
LPS and the low toxicity of MDP-Lys [45
], MDP-Lys is
thought to be a useful immunoadjuvant.
The mechanism by which MDP-Lys activates immune-competent cells remains
unknown. There is growing evidence that pathogen-associated molecules
such as LPS, peptidoglycan, and lipoteichoic acid are recognized by the
specific receptors, including several toll-like receptors (TLRs) and
CD14 [46
47
48
]. Because MDP is a component of the
peptidoglycan of bacterial cell walls, it is possible that these
receptors are involved in the recognition of MDP. Recently, Yang et al.
demonstrated that the activation of MDP on the human monocytic cell
line THP-1 was not inhibited by anti-CD14 or anti-TLR4 mAbs
[49
]. Furthermore, MDP was shown to efficiently
stimulate U937 cells differentiated by an analog of 1,
25-dehydroxyvitamin D3, 22-oxyacalcitriol, which expressed almost no
TLR2 [49
]. These data suggest that MDP exerts its
effects in a CD14-, TLR4-, and TLR-2-independent manner. However, there
is the possibility that other TLRs act as receptors for MDP. It is
interesting that MDP has been shown to interact with serotonin
receptors on macrophages and to enhance superoxide production by them
[50
, 51
]. Further studies will be required
to elucidate the recognition and signaling mechanisms of MDP by DCs.
In conclusion, our findings are the first to clearly indicate that MDP-Lys can activate human DCs to up-regulate the expression of costimulatory molecules and to produce various cytokines, resulting in enhancement of their antigen-presenting function. Thus, the activation of DCs by MDP-Lys is likely to be involved in its immunoadjuvant activities in vivo.
Received September 28, 2000; revised June 16, 2001; accepted June 18, 2001.
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