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Immunology |
and CD40 Ligand Antagonize the Inhibitory Effects of Interleukin 10 on T-Cell Stimulatory Capacity of Dendritic Cells1
Department of Hematology, Oncology, and Immunology, University of Tübingen, D-72076 Tübingen, Germany
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, and CD40 ligation on IL-10
mediated inhibition of DC development and stimulatory capacity. In our
study, the addition of IL-10 to the cultures containing
granulocyte/macrophage-colony stimulating factor and IL-4 with or
without LPS completely inhibited the generation of DCs from peripheral
blood monocytes. These cells remained CD14 positive and expressed high
levels of IL-10 receptor (IL-10R), suggesting that IL-10 mediates its
effects by up-regulating the IL-10R. In contrast, the simultaneous
incubation of monocytes with IL-10 and TNF- or soluble CD40 ligand
(sCD40L) resulted in the generation of CD83-positive DCs, induction of
nuclear localized RelB, and inhibition of IL-10R up-regulation. DCs
grown in the presence of IL-10 and TNF- or sCD40L elicited efficient
CTL responses against viral and tumor-associated peptide antigens,
which, however, were reduced as compared with DC cultures generated
without IL-10. IL-10 decreased the production of IL-6 and the
expression of IL-12 in the presence of TNF- or sCD40L, but it had no
effect on IL-15, IL-18, and TNF- secretion. Our results show that
TNF- or CD40 ligation can antagonize the IL-10-mediated inhibition
on DC function, suggesting that depending on activation stimuli, the
presence of IL-10 does not necessarily result in T-cell anergy. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, IL-1, LPS, CD40 ligation, or by
monocyte-conditioned medium (3
, 9, 10, 11, 12, 13, 14, 15, 16, 17)
.
The capacity of monocytes and DCs to migrate to the sites of
inflammation, where they capture the antigens, and subsequently to the
local lymph nodes is regulated by the expression of different
chemokines and chemokine receptors (18, 19, 20, 21, 22, 23, 24)
. Immature DCs
and monocytes produce inflammatory chemokines including MIP-1,
MCP-1, and MCP-2 and express receptors for inflammatory chemokines such
as CCR1, CCR2, CCR5, CCR6, and CXCR1. In contrast, during the
maturation process, DCs down-regulate the expression of inflammatory
chemokines and their receptors and up-regulate constitutive chemokines
such as TARC, ELC, and the CCR7 receptor.
Recently, it was demonstrated the that function of DCs can be negatively affected by IL-10 (25, 26, 27, 28, 29, 30, 31, 32) . IL-10 is an immunosuppressive and anti-inflammatory cytokine that plays a major role in T helper cell regulation by down-regulation of Th1 responses (33) . The biological functions of IL-10 are mediated by its cell surface receptor (IL-10R), which belongs to the IFN receptor-like subgroup of the cytokine receptor family (34 , 35) . In vitro, it was demonstrated that IL-10 can inhibit the cytokine production and antigen-presenting function of DCs, monocytes, and macrophages (27 , 36 , 37) . In DCs, generated from peripheral blood monocytes using GM-CSF and IL-4, IL-10 inhibits IL-12 production and induces a state of antigen-specific anergy in CD4- and CD8-positive T cells. However, these inhibitory effects of IL-10 on DC function are only observed in immature DCs, whereas mature DCs are completely resistant to the action of IL-10.
Interestingly, IL-10 was also shown to be produced and secreted by tumor cells and may therefore be involved in the defective DC function observed in cancer patients. The production of IL-10 by tumor cells was demonstrated to be one of the mechanisms by which tumor cells can escape from immunological recognition and destruction (38, 39, 40) .
In the present study, we analyzed the effect of TNF- and
sCD40L on the IL-10-mediated inhibition of DC development and function
by adding these cytokines and sCD40L on the first day of the DC culture
initiation. We demonstrate that the addition of TNF- or sCD40L
together with IL-10 to the cultures promoted the differentiation of
mature DCs from peripheral blood monocytes expressing CD83 and RelB,
despite the presence of IL-10. DCs grown in the presence of GM-CSF,
IL-4, IL-10, and TNF- or sCD40L efficiently induced antigen-specific
CTL responses and stimulated allogeneic T cells, although at a reduced
capacity as compared with cultures treated without IL-10. Our data
suggest that: (a) TNF- or sCD40L can antagonize the
inhibitory effect of IL-10 on DC development and function;
(b) DCs generated in the presence of IL-10 and TNF- or
sCD40L have an intermediate activation status; and (c) IL-10
differentially regulates the function of DCs, depending on external
stimuli.
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MATERIALS AND METHODS |
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The following cytokines were obtained from R&D systems (Wiesbaden,
Germany): IL-4 (1000 IU/ml), TNF- (10 ng/ml), and IL-10 (10 ng/ml).
Human recombinant GM-CSF (Leucomax; 100 ng/ml) was from Novartis
(Basel, Switzerland), and LPS was obtained from Sigma (10 µg/ml).
For stimulation of adherent cells with CD40L, we used a soluble recombinant human trimeric CD40 ligand (Biozol, Eiching, Germany; 500 µg/ml). The cultures were fed with fresh medium and cytokines every 2–3 days, and cell differentiation was monitored by light microscopy. The antigen-presenting capacity and expression of cell surface molecules were analyzed after 7 days of culture.
Immunostaining.
Cell staining was performed using FITC- or phycoerythrin-conjugated
mouse mAbs against CD86 and CD40 (all purchased from PharMingen,
Hamburg, Germany); CD3, CD19, CD20, CD80, HLA DR, CD54, and CD14
(Becton Dickinson, Heidelberg, Germany); CD83 (Coulter-Immunotech
Diagnostics, Hamburg, Germany); CD1a (OKT6, Ortho Diagnostic Systems);
and T6-RD1 (Coulter Immunology, Hialeah, FL), and mouse IgG isotype
controls. For the detection of the IL-10R expression, we used an
unconjugated mouse mAb (R&D systems, Wiesbaden, Germany) and stained
the cells afterward with a FITC-labeled goat-antimouse mAb (Becton
Dickinson). Samples were analyzed on a FACScan Calibur (Becton
Dickinson).
Cytokine Determination.
Cytokine concentrations in cell cultures supernatants were measured by
commercially available two-site sandwich ELISAs from R&D systems
(Wiesbaden, Germany; IL-15 and IL-18) or Coulter-Immunotech Diagnostics
(Hamburg, Germany; IL-12, IL-10, IL-6, and TNF-), according to the
manufacturer’s instructions.
MLR Assay.
105 responding cells, either from allogeneic or
autologous PBMNCs, were cultured in 96-well flat-bottomed microplates
(Nunc) with various numbers of irradiated stimulator cells. To use the
same percentage of DCs in the assays, the number of added DCs was
assessed based on fluorescence-activated cell sorter data (cells
expressing CD1a and/or CD83 on the cell surface) and confirmed by
counting of the cells after staining with trypan blue under a light
microscope. Thymidine incorporation was measured on day 5 by a 16-h
pulse with [3
H]thymidine (0.5 µCi/well;
Amersham Life Science, Buckingham, United Kingdom).
Induction of Antigen-specific CTL Response Using an
HLA-A2-restricted Synthetic Peptide.
The IMP 58–66, GILGFVFTL, pol HIV-1 reverse transcriptase peptide
(HIV) 476–484, ILKEPVHGV, and Her-2/neu-derived E75 peptide KIGSFLAFL
(42)
were synthesized using standard Fmoc chemistry on a
peptide synthesizer (432A; Applied Biosystems, Weiterstadt, Germany)
and analyzed by reverse phase high-performance liquid chromatography
and mass spectrometry. For CTL induction, 5 x 105 DCs were pulsed with 50 µg/ml of the
synthetic IMP peptide for 2 h, washed, and incubated with
2.5 x 106 autologous PBMNCs in
RP10 medium. Cells were restimulated after 7 days of culture, and 1
ng/ml human recombinant IL-2 (R & D Systems, Wiesbaden, Germany)
were added every other day (41)
. The cytolytic activity of
induced CTLs was analyzed on day 5 after the last restimulation in a
standard 51Cr-release assay.
CTL Assay.
The standard 51Cr-release assay was performed
with some modifications as described (42)
. Target cells
(T2 cells, 174xCEM.T2 hybridoma, TAP1- and TAP2-deficient), A498 (renal
cell carcinoma, HLA-A2+, Her-2/neu+), K562 (no MHC expression,
sensitive to NK cell-mediated lysis), and SK-OV-3 (ovarian cancer,
HLA-A2 negative, Her-2/neu+) were pulsed with 25 µg/ml peptide for
2 h or left unpulsed and labeled with
[51Cr]sodium chromate in RP10 for 1 h at
37°C. Cells (104
) were transferred to a well of
a round-bottomed, 96-well plate. Varying numbers of CTLs were added to
give a final volume of 200 µl and incubated for 4 h at 37°C.
At the end of the assay, supernatants (50 µl/well) were harvested and
counted in a microbeta counter (Wallac). The percentage of specific
lysis was calculated as: 100 x (experimental
release - spontaneous release/maximal release - spontaneous release). Spontaneous and maximal release were
determined in the presence of either medium or 1% Triton X-100,
respectively.
Preparation of Nuclear Extracts.
Nuclear extracts were prepared from DCs as described previously
(43)
. Briefly, cell pellets were washed in 1 ml of
ice-cold Buffer A [10 mM HEPES (pH 7.9), 1.5
mM MgCl2, 10 mM KCl, 0.5
mM phenylmethylsulfonyl fluoride, and 1 mM
DTT], incubated for 10 min on ice in 1 ml Buffer A + 0.4%
Igepal CA-630 (Sigma, Munich, Germany). Cell membranes thus obtained
were centrifuged at 750 x g for 5 min.
Pellets were resuspended in 200 µl Buffer B [20
mM HEPES (pH 7.9), 0.42 M
NaCl, 1.5 mM MgCl2, 0.2
mM EDTA, 0.5 mM
phenylmethylsulfonyl fluoride, and 1 mM DTT] and
nuclei were mechanically lysed for 2 h at 4°C. Cell debris were
pelleted 15 min at 7500 x g, and supernatant
was recovered and stored at -70°C until use. Proteinase inhibitors
(aprotinin and leupeptin; Sigma, Munich, Germany) were added to buffers
just before use.
PAGE and Western Blotting for Detection of RelB Protein.
Protein concentration of nuclear extracts were determined using a BCA
assay (Pierce, Rockford, IL). Twenty µg of total protein were
separated on 12% polyacrylic amide gel, blotted on polyvinylidene
difluoride membrane, and probed with a polyclonal rat RelB antibody
C-19 (Santa Cruz, CA); bands were visualized by ECL staining (Amersham
Pharmacia, Freiburg, Germany).
RT-PCR.
RT-PCR was performed with some modifications as described recently
(44)
. Total RNA was isolated from cell lysates using
Qiagen RNeasy anion-exchange spin columns (Qiagen GmbH, Hilden,
Germany), according to the instructions of the manufacturer. Five
hundred ng of total RNA were subjected to first-strand cDNA using an
optimized protocol described by Life Technologies, Inc. (SuperScript
Preamplification System; Life Technologies, Inc., Eggenstein, Germany),
using oligo(dT) as primer. Two µl of cDNA obtained from the reverse
transcriptase reaction were subjected to the amplification. To control
the integrity of the isolated RNA, 1 µl of cDNA was amplified by an
intron-spanning primer pair for the 18S rRNA gene. Primer sequences
were deduced from published cDNA sequences: 28S rRNA,
5'-ACTTAGAGGCGTTCAGTCATAATC-3' and 5'-AGACAGGTTAGTTTTACCCTACTG; MCP-4,
5'-AAATGACTTTTCCATTCTCCTCTG-3' and 5'-TGCATTCATCTTTCCACAATAAAC-3'; and
CCR7, 5'-TGAGCTCAGTAAGCAACTCAACAT-3' and
5'-TAGCTTATCAGCCCTGTCTTTTTC-3'. Primer sequences for IL-12 and DC-CK1
(14)
, MIP-1, MCP-2, ELC, TARC (18)
, and
RelB were published recently. Reactions were amplified in a DNA thermal
cycler (GeneAmp PCR System 2400; Perkin-Elmer). The temperature
profiles were as follows: 5 min at 94°C pretreatment; 60°C for
30 s annealing for the DC-CK1, MCP-4, CCR7, and IL-10R; and 55°C
for the 28S rRNA primers; 72°C for 30 s synthesis; and 94°C
for 30 s denaturation. Finally, a single posttreatment was
performed at 72°C for 5 min. Ten µl of the RT-PCR products were
electrophoresed through a 3% agarose gel and stained with ethidium
bromide for visualization under UV light. Numbers of cycles performed:
15 for 28S rRNA; 30 for IL-12; 32 for RelB; 28 for DC-CK1; 30 for
MIP-1; 32 for MCP-2; 30 for MCP-4; 32 for ELC 32; 30 for CCR7 30;
and 28 for TARC.
Statistical Analysis.
Each experiment was performed at least three times. Representative
experiments are shown. Student’s t test was performed to
evaluate the significance of the results.
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RESULTS |
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or CD40 Ligation but not by LPS., or the soluble trimeric human CD40L (sCD40L) were added
together with the above-mentioned cytokines.
As demonstrated in Fig. 1
, the addition of IL-10 to the cultures together with GM-CSF and IL-4
abolished the differentiation of monocytes to DCs, because the cells
still expressed CD14 and were negative for CD1a and CD83 at day 7 of
culture. Most of the cells remained adherent with a phenotype of
macrophages. When TNF- or the trimeric human sCD40L were added to
the cultures together with GM-CSF, IL-4, and IL-10, the cells developed
into DCs expressing high levels of CD83, HLA-DR, and costimulatory
molecules, comparable with the phenotype of DCs generated in the
absence of IL-10. Interestingly, in cultures incubated with LPS, IL-10
completely inhibited the generation of DCs. Similar results were
obtained when peripheral blood monocytes were preincubated for 1, 2, 3,
or 5 days with GM-CSF and IL-4 and IL-10, LPS, or TNF- or sCD40L
were added for the remaining 6, 5, 4, or 2 days of culture,
respectively (data not shown). The inhibitory effect of IL-10 on DC
development was prevented by the simultaneous addition of a
neutralizing anti-IL-10 antibody (20 µg/ml; R&D Systems, Wiesbaden,
Germany) to the culture medium (data not shown).
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and sCD40L on induction of CD83
expression and up-regulation of costimulatory molecules was already
detectable after 2–3 days of culture (data not shown).
The IL-10 Induced Up-Regulation of IL-10R Expression Is Inhibited
by TNF- or sCD40L.
We used a monoclonal antibody specific for the human IL-10R to analyze
its expression on cells generated from peripheral blood monocytes. As
shown in Fig. 1
, immature DCs generated in the presence of GM-CSF and
IL-4 expressed low levels of IL-10R on the cell surface, whereas mature
DCs cultured with sCD40L or TNF- expressed no IL-10R. The addition
of IL-10 to the cultures containing GM-CSF and IL-4 resulted in a
marked increase of the IL-10R expression, thus suggesting that IL-10
mediates its inhibitory effects by up-regulation of its own receptor.
The presence of CD40L or TNF- inhibited the up-regulation of the
IL-10R on the cell surface by IL-10, which resulted in the development
of DCs with a mature phenotype. In contrast, addition of LPS to
IL-10-treated cultures could not prevent the IL-10R up-regulation (Fig. 1
).
RelB Expression in Cells Generated from Peripheral Blood Monocytes.
RelB belongs to the family of transcription factors associated with the
development and effective antigen presentation of DCs
(45, 46, 47, 48)
. We therefore analyzed RelB expression in
different cell populations generated in vitro from
peripheral blood monocytes by RT-PCR and Western blot. The RelB mRNA
(Fig. 2
) and nuclear localized protein expression in DCs (Fig. 3
) were increased by incubation of the adherent cells with sCD40L, LPS,
or TNF-, whereas no RelB expression was detected in cultures grown
in the presence of GM-CSF and IL-4 with or without IL-10. The
up-regulation of RelB was not inhibited by the addition of IL-10 in the
presence of sCD40L or TNF-, in contrast to the cells generated with
GM-CSF, IL-4, and LPS. These data are in line with the results obtained
from the set of previous experiments (Fig. 1
), thus confirming the
important role of RelB induction for DC development and demonstrating
that the inhibitory effects mediated by IL-10 can be antagonized by
TNF- or sCD40L but not by LPS, and that bacterial and T-cell induced
activation of DC follows distinct pathways.
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or sCD40L.
, the cells grown in the presence of IL-10, GM-CSF, and IL-4 with or
without LPS have a reduced ability to stimulate allogeneic PBMNCs,
whereas the IL-10 treatment had only a moderate inhibitory effect on
the stimulatory capacity of the mature DCs generated with TNF- or
CD40L to trigger the proliferation of allogeneic PBMNCs.
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, CTL lines obtained after two weekly restimulations demonstrated
high peptide-specific killing when DCs were grown in the presence of
TNF-, LPS, or soluble CD40L. The in vitro induced T cells
only elicited a cytotoxic response against targets coated with the
cognate HLA-A2 binding peptide derived from IMP, but they did not
recognize targets coated with an irrelevant HIV-peptide. IL-10
completely inhibited the stimulatory capacity of cells generated in
cultures containing GM-CSF and IL-4 (Fig. 4A
). In contrast,
DCs grown in cultures supplemented with IL-10 and TNF- or soluble
CD40L induced an efficient peptide-specific CTL response, which,
however, was reduced as compared with cells generated without IL-10
(Fig. 4 and C
). CTLs induced with cells
generated in presence of IL-10 and LPS did not induce any measurable
antigen-specific cytotoxic activity, confirming again that LPS in
contrast to TNF- or sCD40L cannot antagonize the inhibitory effects
of IL-10 (Fig. 4D
).
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(Fig. 5 and D
) or soluble CD40L (Fig. 5 and F
) to induce a primary CTL response against a
tumor-associated antigen, cells were pulsed with the E75 peptide
derived from the Her-2/neu antigen (42)
. As demonstrated
in Fig. 5
, the CTLs recognized target cells pulsed with the E75 peptide
in an antigen-specific manner. Furthermore, the E75-specific CTLs
efficiently lysed tumor cells expressing Her-2/neu (A498 cell line),
whereas they did not recognize Her-2/neu+/HLA-A2 cells (SK-OV-3 cells)
or the NK cell-sensitive K562 tumor cells, confirming that the
cytotoxic activity of these CTLs is antigen specific and HLA-restricted
and demonstrating that TNF- or soluble CD40L can prevent the
recently described IL-10-mediated inhibition of antigen-specific CTL
responses. However, in line with the previous results, the cytotoxic
activity of these antigen-specific CTLs was reduced as compared with
the untreated DC cultures.
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or CD40L with or without IL-10
seem to have a similar phenotype, but they differ in their T-cell
stimulatory function. We therefore additionally analyzed cytokine
production and expression of various chemokines by the different cell
populations.
Cytokine Production by Cultured Cells.
The T-cell stimulatory capacity of DCs is regulated by the expression
of MHC, costimulatory and adhesion molecules on the cell surface, and
the production of cytokines like IL-12, IL-15, IL-18, TNF-, or IL-6
that are important for the induction of Th1 responses and proliferation
of naive T-cells (14
, 49, 50, 51, 52, 53, 54, 55, 56, 57)
. Cytokine secretion by
generated cells in vitro was analyzed using commercially
available two-site sandwich ELISAs. As shown in Table 2
, IL-10 treatment of cell cultures containing GM-CSF and IL-4 resulted
in decreased levels of cytokine production of the cells. However, when
the cells were grown in the presence of sCD40L or TNF- together with
GM-CSF, IL-4, and IL-10, the supernatants contained similar levels of
IL-15, IL-18, and TNF- as compared with the cultures without IL-10.
In contrast to these results, sCD40L or TNF- could not overcome the
inhibition of the IL-6 by IL-10. Analysis of IL-12 (p40) expression
using RT-PCR (Fig. 2
) demonstrated that IL-10 down-regulated the IL-12
expression in all cell populations, even in the presence of sCD40L or
TNF-, similar to a previous report where IL-10 inhibited IL-12
production of CD40 ligand-activated immature DCs (27)
. In
line with these RT-PCR results, the presence of IL-10 inhibited
completely the IL-12 production (p70 heterodimer) in the cultures
independent of the stimuli used for activation. sCD40L or TNF- lead
to very low levels of bioactive IL-12 secretion, consistent with
previous reports (58
, 59)
. Higher levels of IL-12 were
detectable after LPS stimulation.
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and MCP-2) and
constitutive chemokines like DC-CK1, TARC, and ELC (an EBI1-ligand
chemokine) as well as the corresponding receptors that underline the
ability of DCs to attract other cells as well as to regulate their own
function and migratory capacity (18, 19, 20, 21, 22, 23, 24
, 60)
.
As shown in Fig. 6
using RT-PCR, DC-CK1 mRNA was found as expected in immature DCs
generated with GM-CSF and IL-4. As demonstrated above (Fig. 1
),
addition of IL-10 to these cultures prevented the generation of DCs
from adherent monocytes, and the cells retained the monocyte/macrophage
phenotype. Interestingly, these cells nevertheless expressed DC-CK1
(Fig. 6
), in contrast to freshly purified peripheral blood monocytes
(data not shown; Ref. 14
). Incubation of the cells with
CD40L or TNF- resulted in down-regulation of the DC-CK1 mRNA in DCs.
This effect of sCD40L and TNF- was inhibited by addition of IL-10.
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, all DCs generated in the presence of sCD40L
or TNF- with or without IL-10 expressed ELC, TARC, and the CCR7
receptor. Interestingly, maturing DCs generated in the medium
containing IL-10 expressed higher levels of the inflammatory chemokines
MIP-1 and MCP-2.
Using subtractive cDNA libraries generated from DCs and peripheral
blood monocytes, we recently detected a selective MCP-4 mRNA
(61)
expression in
DCs.4
As demonstrated in Fig. 6
, IL-10 up-regulated the expression of MCP-4
by DCs, even in the presence of sCD40L and TNF-. We also found MCP-4
transcripts in cells incubated with GM-CSF, IL-4, and IL-10, whereas we
were not able to detect MCP-4 transcripts in freshly isolated blood
monocytes (data not shown).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Previous studies demonstrated that DCs can develop in vitro
from CD14+ blood monocytes cultured with GM-CSF and IL-4. These cells
have the phenotypic and functional characteristics of immature DCs and
can be further induced to mature by activation with TNF-, by CD40
ligation, monocyte-conditioned medium, LPS, or IL-1
(10, 11, 12, 13, 14)
. Several recent reports demonstrated that IL-10
can inhibit the differentiation and function of immature DCs generated
from peripheral blood monocytes, whereas mature DCs are resistant to
its action (25, 26, 27, 28, 29, 30, 31, 32)
. Monocytes are recognized as an
alternative source of DC precursors. They migrate to the sites of
inflammation attracted by cytokines and chemokines released. Upon
contact with cytokines like GM-CSF, IL-4, TNF-, LPS, T cells, or
even IL-10, they may differentiate into DCs. Therefore, in contrast to
the in vitro culture conditions, the differentiation of
monocytes toward DCs in vivo is influenced by a combined
action of different cytokines.
In our study, we therefore analyzed the combined influence of various
stimuli like TNF-, sCD40L, LPS, and IL-10 on the generation and
function of DCs from adherent peripheral blood monocytes using GM-CSF
and IL-4 by addition of these cytokines at the time of culture
initiation. In line with previous results, we show that IL-10 prevents
the differentiation of DCs when IL-10, GM-CSF, and IL-4 are added to
the medium on the first day of the culture initiation. However, the
simultaneous addition of TNF- or soluble CD40 ligand, together with
IL-10, resulted in generation of CD83-positive DCs that displayed the
phenotype of mature DCs and expressed high levels of costimulatory and
MHC molecules, suggesting that TNF- or CD40 ligation can antagonize
the inhibitory effect of IL-10 on DC development. Interestingly, LPS
could not antagonize the inhibitory effects of IL-10 on DC development,
suggesting that bacterial and T cell-induced activation of DCs follows
different pathways.
The inhibitory effect on the DC generation correlated with the level of the IL-10R expression. Addition of IL-10 to the cultures containing GM-CSF and IL-4 with or without LPS resulted in the up-regulation of the IL-10R on the cell surface. These results might explain why mature DCs, in contrast to immature DCs, are resistant to the action of IL-10.
Our results are complementary to the recent report by Buelens et al. (27) . Using immature DCs after a 7-day culture with GM-CSF and IL-4, they demonstrated that CD40 ligation can, in contrast to LPS, induce maturation of DCs in the presence of IL-10 characterized by expression of CD83 and up-regulation of costimulatory molecules.
In addition to the phenotypic analysis, we investigated the functional properties of the various cell populations generated with or without IL-10 in terms of RelB induction, cytokine production, expression of chemokines, and initiation of antigen-specific CTL responses.
The transcription factor RelB is a member of nuclear factor-
B/Rel
family. In several reports, it was demonstrated that RelB is essential
for the development of myeloid-related DCs and their
antigen-presenting function (45, 46, 47, 48)
. In line with the
results from the phenotypic studies, IL-10 had no effect on mRNA or
nuclear protein expression of RelB in the presence of TNF- or
sCD40L, whereas LPS failed to overcome the inhibition mediated by IL-10
(Fig. 3
), confirming the important role of RelB expression for the
development and function of DCs.
Functional analyses of the phenotypically mature DCs generated with
TNF- or sCD40L revealed that these cells were potent stimulatory
cells, even in the presence of IL-10, and induced primary
antigen-specific CTLs that were able to lyse tumor cells expressing the
corresponding tumor-associated antigen, thus demonstrating that sCD40L
or TNF- can antagonize the suppression of antigen-specific T-cell
responses mediated by IL-10. However, the addition of IL-10 to the
cultures resulted in a reduced capacity of these DCs to stimulate
allogeneic T cells in a MLR and to induce antigen-specific MHC class
I-restricted cytotoxic T cells directed against viral and
tumor-associated antigens as compared with cultures without IL-10
(Figs. 4
5
; Table 1
).
This observation is of special interest, because IL-10 has been shown to be produced and secreted by tumor cells and may therefore be involved in the defective DC function observed in cancer patients. Furthermore, the production of IL-10 by tumor cells was demonstrated to be one of the mechanisms by which tumor cells can escape from immunological recognition and destruction (38, 39, 40) .
Analysis of cytokine secretion in the cell cultures demonstrated that
DCs grown with IL-10 and TNF- or soluble CD40L expressed lower
levels of IL-6 and IL-12 as compared with cells generated without
IL-10. The reduced capacity to secrete IL-6, a factor known to
costimulate the proliferation of naive Th cells (57)
, and
the reduced IL-12 expression may be one possible explanation for their
reduced functional APC properties, because we could not detect
significant differences in the production of IL-15, IL-18, or TNF-,
and other factors may also contribute to this phenomenon.
DCs generated in vitro using GM-CSF, IL-4, and TNF- or
sCD40L with or without IL-10 expressed TARC and ELC and the CCR7
receptor, which was shown to be important for the migration of DCs from
tissues to the lymphatic vessels and nodes and initiation of T-cell
activation (18
, 22, 23, 24)
. The presence of IL-10 resulted in
up-regulation of the inflammatory chemokines MIP-1, MCP-2, and
MCP-4, which are responsible for attracting cells involved in
inflammatory reactions and are produced at early time points of DC
activation, suggesting that these DCs have a less mature phenotype,
although they express CD83 and high levels of costimulatory molecules.
DC-CK-1 is a recently identified chemokine that is selectively
expressed in DCs with preferential chemotactic activity for naive T
cells (14
, 58)
. Surprisingly, DC-CK1 mRNA expression was
up-regulated in mature DCs treated with IL-10 and down-regulated in the
absence of IL-10 (Fig. 6
). In cells incubated with GM-CSF, IL-4, and
IL-10, displaying the phenotype of monocytes/macrophages and lacking
any stimulatory capacity as analyzed by MLR and CTL induction, a high
level of DC-CK-1 expression was detected by RT-PCR. Expression
of DC-CK-1 could not be found in purified, freshly isolated
CD14-positive peripheral blood monocytes (14)
. Thus,
monocytes or macrophages can express DC-CK-1 upon contact with IL-10
and attract naive T cells without their further activation, which might
lead to anergy.
In conclusion, phenotypically mature DCs generated in vitro
in the presence of IL-10 and TNF- or sCD40L seem to have an
intermediate activation status that is modulated by the effect of
IL-10. These data support the concept of the regulatory function
mediated by IL-10 during inflammatory reactions and induction of immune
responses. Furthermore, our results demonstrate that, depending on
stimuli provided by T cells or bacterial components during DC
development, the presence of IL-10 does not necessarily result in
induction of T-cell anergy, and sCD40L or TNF- can prevent the
IL-10-mediated inhibition of antigen-specific CTL induction by DCs.
These results might be important for development of efficient
immunotherapies designed to overcome the tumor-mediated
immunosuppression because IL-10 was shown to be an important factor
suppressing APC function in cancer patients.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported in part by Grant SFB 510
from the Deutsche Forschungsgemeinschaft and grants from the Deutsche
Krebshilfe and the fortune Programm of the
University of Tübingen. 
2 To whom requests for reprints should be
addressed, at Department of Hematology, Oncology, and Immunology,
University of Tübingen, Otfried-Müller-Strasse-10, D-72076
Tübingen, Germany. Phone: 49-70712982726; Fax: 49-7071293671;
E-mail: peter.brossart{at}med.uni-tuebingen.de
3 The abbreviations used are: DC, dendritic cell;
IL, interleukin; IL-10R, IL-10 receptor; TNF, tumor necrosis factor;
LPS, lipopolysaccharide; sCD40L, soluble CD40 ligand; GM-CSF,
granulocyte/macrophage-colony stimulating factor; PBMNC, peripheral
blood mononuclear cell; mAb, monoclonal antibody; IMP, influenza matrix
protein; RT-PCR, reverse transcription-PCR; MLR, mixed lymphocyte
reaction; APC, antigen-presenting cell; TARC, thymus and
activation-regulated chemokine; NK, natural killer.
Received 2/ 3/00. Accepted 6/16/00.
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