| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
-Induced Activation and Maturation of Human Peripheral Blood Monocyte-Derived Dendritic Cells
*
Department of Cell Processing, Institute of Medical Science, University of Tokyo, Tokyo, Japan; and
Japanese Red Cross Central Blood Center, Tokyo, Japan
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
or IL-10 has been implicated to reversibly regulate
physiological states of dendritic cells (DCs). However, little is known
about dual stimulations of these cytokines on DC properties and the
intracellular signaling events that are responsible for the regulation
of these states. Here, we show that a family of mitogen-activated
protein kinases (MAPKs), extracellular signal-regulated kinase 2
(ERK2), stress-activated protein kinase/c-Jun N-terminal kinase
(SAPK/JNK), and p38mapk, are potentially
involved in IL-10-mediated selective suppression of TNF--induced
changes of the monocyte-derived DC properties. TNF- induced the
cluster formation of the cells and the enhancement of cell surface
expression levels of CD83, CD86, and HLA-DR, and T cell stimulatory
capacity, whereas the capacities for the endocytosis and the
chemotactic migration were suppressed in these cells. Treatment of
monocyte-derived DCs with IL-10 resulted in the reduction of the cell
surface expression levels of CD86, HLA-DR, and T cell stimulatory
capacity, whereas both endocytic and chemotactic migratory capacities
were increased by IL-10. Dual stimulations of monocyte-derived DCs with
TNF- and IL-10 selectively antagonized their respective effects on
these DC properties. TNF- induced tyrosine phosphorylation and
enzymatic activation of ERK2, SAPK/JNK, and
p38mapk, whereas IL-10 did not induce
these events. Dual stimulations of TNF- plus IL-10 abolished
TNF--induced changes of these MAPKs in DCs. These results suggest
that the blockage in the MAPKs cascades contributes to IL-10-mediated
repression of TNF--induced changes of DC
properties. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The characterization of DCs is difficult because they represent only a
small subpopulation that includes interdigitating reticulum cells in
lymphoid organs, blood DCs, Langerhans cells in the epidermis of the
skin, and dermal DCs (1). Recently, an in vitro culture system that
enables progenitors in peripheral blood, bone marrow, and cord blood to
differentiate into DCs has been established, and it has revealed the
basic mechanisms underlying the properties of DCs (7, 8, 9, 10). Previous
studies have shown that TNF- promotes activation and maturation of
DCs, whereas IL-10 suppresses several DC properties in vitro (8, 9, 11, 12, 13, 14, 15, 16). However, little is known about the effect of dual stimulations
of TNF- and IL-10 on morphological, phenotypical, and functional
states of DCs.
TNF- secreted by activated T cells, monocytes/macrophages, and DCs
is a pleiotropic cytokine that has growth modulatory, cytotoxic, and
inflammatory activities (14, 17). The effects of TNF- are mediated
by two distinct cell surface receptors of 
55 kDa (TNF-R1, CD120a)
and 75 kDa (TNF-R2, CD120b) (18), and TNF-R1 is involved in
TNF--induced phenotypical and functional changes in DCs (10). TNF-R1
receptor engagement by TNF- initiates complex signaling events,
including protein tyrosine kinase (PTK)-dependent cascades and a
ceramide-mediated pathway leading to the activation of two
transcription factors, NF-
B and activator protein 1, which regulate
the expressions of numerous immune/inflammatory response genes
(18).
IL-10, which is a cytokine produced by activated T cells, B
cells, keratinocytes, monocytes/macrophages, and DCs, suppresses
cytokine synthesis by activated T cells, NK cells,
monocytes/macrophages, and DCs (15, 16). IL-10 can also block the
ability of monocytes/macrophages and DCs to act as APCs via
down-regulation of MHC products and costimulatory molecules (15, 16).
The biological functions of IL-10 are mediated through the cell surface
receptor IL-10R, which is a member of the IFN receptor-like subgroup of
the cytokine receptor family (19, 20). Stimulation with IL-10 results
in tyrosine phosphorylation and activation of the Janus kinase (JAK)
family, including JAK1, Tyk2, and their effectors, and the STAT,
STAT1 and STAT3 (21). Recent studies have shown that IL-10 induces
activation of phosphatidylinositol 3-kinase (PI-3 kinase) and p70 S6
kinase (22). However, these intracellular events do not appear to be
responsible for the suppressive effects of IL-10 on immune/inflammatory
responses, and the signal transduction events causing these effects
remain unclear (21, 22).
Mitogen-activated protein kinases (MAPKs) are activated following
engagement of a variety of cell surface receptors via dual tyrosine and
threonine phosphorylation and are thought to be involved in various
cellular responses (23, 24, 25, 26). The various members of the MAPK families
differ in their substrate specificity and are activated by distinct
upstream regulators and extracellular stimuli (23, 24, 25, 26). Currently, the
MAPK family is comprised of three subfamilies, namely: 1) the
extracellular signal-related kinase (ERK) subfamily, including
p44mapk/erk1 (ERK1) and
p42mapk/erk2 (ERK2); 2) the
stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK)
subfamily, including the p46 SAPK/JNK and p54 SAPK/JNK isoforms and
their variants; and 3) the p38mapk
subfamily (23, 24, 25, 26). Previous studies have shown that ligation of
TNF- with TNF-R1 initiates activation of ERK2, SAPK/JNK, and
p38mapk in several cells and cell lines
(23, 24, 25, 26). On the other hand, stimulation with IL-10 does not activate
the p21ras/Raf/ERK2 pathway in human
monocytes and murine cell lines (20, 27). However, much less is known
about the intracellular signaling that are responsible for mediating
the TNF- and IL-10-regulated DC properties.
Here, we show that TNF- and IL-10 exhibit the opposite effects on
morphological, phenotypical, and functional states of human peripheral
blood monocyte-derived DCs, and dual stimulations of these cells with
these cytokines antagonized their respective effects on several DC
properties. Furthermore, stimulation of DCs with TNF- induced
tyrosine phosphorylation and activation of ERK2,
p38mapk, and SAPK/JNK, whereas IL-10 failed
to induce this activation. Dual stimulations with TNF- and IL-10
suppressed TNF--induced modulation of these MAPKs. Our results
suggest that the repression of MAPK cascade may be crucially involved
in IL-10-mediated negative regulation of TNF--induced changes of
human peripheral blood monocyte-derived DC properties.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The medium used was RPMI 1640 supplemented with 2 mM
L-glutamine, 50 µg/ml streptomycin, 50 U/ml penicillin,
and 10% heat-inactivated FCS. Granulocyte-macrophage CSF (GM-CSF) was
kindly provided by Kirin Brewery (Tokyo, Japan). IL-4, IL-10, TNF-,
and RANTES were purchased from PeproTech (London, U.K.). FITC-labeled
dextran (FITC-DX) and lucifer yellow (LY) were purchased from Molecular
Probes (Eugene, OR). Horseradish peroxidase (HRP)-conjugated
anti-phosphotyrosine mAbs (clone RC20) were purchased from
Transduction Laboratories (Lexington, KY). ERK2,
p38mapk and SAPK/JNK immunoblotting kits
and their kinase assay kits were purchased from New England Biolabs
(Beverly, MA). A mAb to IL-10 was purchased from PharMingen (San Diego,
CA).
In vitro generation and culture of human DCs
DCs were generated from PBMC, as described previously (8, 9, 10),
with some modification (28). Briefly, PBMC were obtained from 30 ml of
leukocyte-enriched buffy coat from healthy donors by centrifugation
with Ficoll-Hypaque (Pharmacia, Uppsala, Sweden), and the light density
fraction from the 42.5–50% interface was recovered. The cells were
resuspended in culture medium and allowed to adhere to 6-well plates
(Costar, Cambridge, MA). After 2 h at 37°C, nonadherent cells
were removed, and adherent cells (90% CD14+ cells) were
cultured in 3 ml of medium supplemented with GM-CSF (50 ng/ml) and IL-4
(250 ng/ml). After 7 days of culture, DCs were harvested, washed, and
subsequently cultured in the presence or absence of TNF- (50 ng/ml),
IL-10 (50 ng/ml), or a mixture of TNF- and IL-10 for an
additional 4 days. The resulting cells were used for subsequent
experiments. These cell populations have the typical dendritic
morphology and phenotype (>85% CD1a+ cells; >95%
CD11c+ cells; <1% CD14- cells;
>75% CD86+ cells;> 85% HLA-DR+ cells). The
cell morphology was monitored by light microscopy.
Isolation of T cells from PBMC
T cells were prepared using a T cell-enrichment immunocolumn (Biotex Laboratories, Edmonton, Canada) from leukocyte-enriched buffy coat as, described above. T cell preparations were typically >90% pure, as indicated by anti-CD3 mAb (Becton Dickinson, Mountain View, CA) staining.
Flow cytometry
For surface marker analysis, DCs were cultured with one of the following mAbs conjugated to phycoerythin (PE) for direct fluorescence: CD83 (Coulter Immunology, Hialeah, FL), CD86 (PharMingen), and HLA-DR (Becton Dickinson). Cells were also stained with the corresponding PE-conjugated isotype-matched control mAb (Becton Dickinson). Thereafter, the cells were washed twice and suspended in PBS containing 0.2 µg/ml propidium iodide (Sigma, St. Louis, MO) to exclude dead cells. Analysis of fluorescence staining was performed with a FACSCalibur flow cytometer (Becton Dickinson) and CELLQuest Software.
Endocytosis assay with FITC-DX and LY
The methods used to determine the endocytotic activity of in vitro-generated DCs have previously been described (10). Briefly, FITC-DX or LY was added to a final concentration of 1 mg/ml to the cells, and the cells were cultured for 60 min at 37°C. After incubation, cells were washed four times with ice-cold PBS and analyzed by flow cytometry, as described above.
Assay for chemotaxis
The in vitro migration of cells was assessed in a Transwell cell culture chamber (Costar) as described previously (28). In brief, polycarbonate filters with 8.0-µm pore size were precoated with 5 µg of gelatin in a volume of 50 µl on the lower surface and dried overnight at room temperature. The coated filters were washed in PBS and then dried immediately before use. DCs (106/100 µl) were added to the upper compartment of the chamber. RANTES diluted in serum-free culture medium (1–100 ng/ml) was loaded in the lower compartment. After a 2-h incubation, the filters were fixed with methanol and stained with hematoxylin and eosin. The cells on the upper surface of the filters were removed by wiping with cotton swabs. The cells that had migrated to various areas of the lower surface were manually counted under a microscope at a magnification of x400, and each assay was performed in triplicate. The data were expressed as number (no.) of migrated cells/field.
MLR
Responding T cells (105) from an unrelated individual (allogeneic MLR) were cultured in 96-well flat-bottom microplates (Costar) with different numbers (102-5 x 104) of irradiated (15 Gy from a 137Cs source) stimulator cells. Thymidine incorporation was measured on day 5 by an 18-h pulse with 0.5 µCi/well of [3H]thymidine (1 µCi/well; sp. act., 5 Ci/mmol) (Amersham Life Science, Buckinghamshire, U.K.).
Immunoblotting
Monocyte-derived DCs (106) were starved in
serum-free medium for 24 h at 37°C and subsequently kept for
4 h on ice to reduce the basal level of tyrosine phosphorylation
of intracellular proteins. The cells were either unstimulated or
incubated with TNF- (50 ng/ml), IL-10 (50 ng/ml), or a mixture of
TNF- and IL-10 in the presence or absence of anti-IL-10 mAb (10
µg/ml) for 5 min at 37°C. The cells were washed twice in cold PBS
and resuspended in 100 µl of lysis buffer (1% Nonidet P-40, 20 mM
Tris-HCl (pH 8.0), 137 mM NaCl, 10% glycerol, 2 mM EDTA, 10 µg/ml
leupeptin, 10 µg/ml aprotinin, 1 mM PMSF, and 1 mM sodium
orthovanadate). The nuclei and the insoluble cell debris were removed
by centrifugation at 4°C for 10 min at 14,000 g. The
postnuclear extracts were collected and used as total cell lysates.
Total cell lysates or the immunoprecipitates were suspended in 2x SDS
sample buffer (313 mM Tris-HCl (pH 6.8), 10% SDS, 2-ME, 50% glycerol,
and 0.01% bromphenol blue) and heated at 95°C for 3 min. The protein
samples were fractionated by 12% SDS-PAGE and transferred onto
polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA).
The nonspecific Ab binding sites on the membrane were blocked with 1%
BSA, 0.1% Tween 20 in saline (10 mM Tris-HCl (pH 7.4), 100 mM NaCl)
for 20 min at 37°C. The membrane was then incubated for 16 h at
4°C with HRP-conjugated anti-phosphotyrosine mAb and washed for
15 min with 0.5% Tween 20 in saline. Immunoblotting of ERK2, SAPK/JNK,
or p38mapk were performed according to the
instruction manual. Briefly, the membranes were incubated for 16 h
at 4°C with mAbs to anti-tyrosine phosphorylated ERK2, SAPK/JNK,
or p38mapk, washed for 15 min, and
incubated with HRP-conjugated secondary Abs for 1 h at room
temperature. Blots were visualized by enhanced chemiluminescence (ECL;
New England Biolabs). To ensure similar amounts of MAPKs in each
sample, the same membrane was stripped off, reprobed with mAbs to ERK2,
SAPK/JNK, or p38mapk, and developed with
HRP-conjugated secondary Abs by ECL.
Immune complex kinase assay
Kinase activities of ERK2, SAPK/JNK, or
p38mapk were determined according to the
instruction manual. In brief, monocyte-derived DCs (4 x
106) were either unstimulated or incubated with TNF- (50
ng/ml), IL-10 (50 ng/ml), or a mixture of TNF- and IL-10 for 5 min
at 37°C. The cells were subsequently lysed with 100 µl of lysis
buffer (1% Triton X-100, 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM
EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 mM ß-glycerol
phosphate, 1 mM sodium orthovanadate, 1 µg/ml leupeptin), and the
lysates were subjected to immunoprecipitation with anti-tyrosine
phosphorylated ERK2 mAb (for ERK kinase activity), c-Jun fusion protein
(for SAPK/JNK kinase activity), or anti-tyrosine phosphorylated
p38mapk mAb (for
p38mapk kinase activity), followed by
protein G-Sepharose 4 fast flow (Pharmacia). The complexes were washed
three times with lysis buffer and twice with kinase buffer (25 mM
Tris-HCl (pH 7.5), 5 mM ß-glycerol phosphate, 2 mM DTT, 0.1 mM sodium
orthovanadate, and 10 mM MgCl2) before resuspending in 20
µl of kinase buffer containing 100 µM ATP, 1 µg of Elk-1 (for ERK
kinase activity), or activating transcription factor (ATF)-2
(for p38mapk kinase activity). The mixtures
were incubated at 30°C for 30 min. Reactions were terminated with the
addition of 20 µl of 2x SDS sample buffer. Sample were heated at
95°C for 3 min, separated by 12% SDS-PAGE, and transferred onto PVDF
membranes. The membranes were probed with mAbs to phospho-Elk-1 (for
ERK kinase activity), -c-Jun (for SAPK/JNK kinase activity) or -ATF-2
(for p38mapk kinase activity), and
developed with HRP-conjugated secondary Abs by ECL.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and IL-10 reversibly regulate phenotypic changes of
monocyte-derived DCsThe physiological states of DCs are believed to be strictly regulated by extracellular stimuli, including cytokines, adhesion/costimulatory molecules, and bacterial products. The activation and maturation states of DCs are correlated with morphological, phenotypical, and functional changes including up-regulation and down-regulation of cell surface expression levels of MHC products and adhesion/costimulatory molecules, migratory capacity, Ag uptake, and processing capacity, and T cell stimulatory capacity.
Previous studies have shown that TNF- promotes activation and
maturation states of DCs, whereas IL-10 suppresses several DC
properties (8, 9, 11, 12, 13, 14, 15, 16). However, little is known about the effect of
dual stimulations of TNF- and IL-10 on DC properties and the
intracellular signaling responsible for mediating these states
following these cytokine exposures. In an attempt to clarify the effect
of dual stimulations of TNF- and IL-10 on DC properties, GM-CSF plus
IL-4-generated human peripheral blood monocyte-derived immature DCs
were cultured with or without TNF- (50 ng/ml), IL-10 (50 ng/ml), or
a mixture of TNF- and IL-10 for 4 days, and morphological,
phenotypical, and functional analyses of the resulting cells were
performed. It has been previously shown that the expression levels of
CD86 and HLA-DR increase during activation and maturation of DCs (8, 11, 12, 13, 14), and CD83 has been identified as a selective marker of mature
DCs (9). Fig. 1
shows that treatment of monocyte-derived
DCs with TNF- resulted in increased expression of CD86 and HLA-DR,
and CD83 was induced on the cell surface, whereas cell surface
expression levels of CD86 and HLA-DR were inhibited by IL-10 when
compared with unstimulated cells. On the other hand, the combination of
TNF- and IL-10 antagonized their respective effects on cell surface
expression levels of CD86 and HLA-DR. Interestingly, IL-10 did not
affect TNF--induced cell surface expression of CD83.
|
). Light microscopy revealed that treatment of the
cells with TNF- results in the formation of homotypic aggregates,
whereas IL-10 failed to induce these events, indicating that maturation
of monocyte-derived DCs is also associated with their cluster
formation. We also found that IL-10 had little or no effect on
TNF--induced aggregation of these cells. These results indicate that
IL-10 selectively abrogates TNF--induced changes of properties of
monocyte-derived DCs in terms of morphologic and phenotypic features.
|
and IL-10 antagonized their respective
effects on the functional properties of monocyte-derived DCs
Immature DCs capture and process Ags via their high endocytic
capacity, and they lose their endocytic/processing activities of Ags
and mature into potent immunostimulatory APCs during differentiation
(6). Previous studies have shown that the endocytic capacity of DCs was
suppressed by TNF- during their maturation process (8, 10), whereas
IL-10 enhanced this function (16). To assess the effect of dual
stimulations of TNF- and IL-10 on the endocytic capacity of DCs,
mannose receptor-mediated endocytosis of FITC-DX and
macropinocytosis of LY via a cytoskeleton-dependent type of fluid-phase
endocytosis were monitored. As shown in Fig. 3A, TNF- inhibited the uptake of FITC-DX
and LY and their accumulation into DCs, whereas IL-10 increased the
endocytosis of these molecules in these cells compared with those of
unstimulated cells. On the other hand, comparable results were observed
using the combination of TNF- and IL-10 when compared with
unstimulated immature DCs.
|
induced down-regulation of chemotactic
migratory capacity in response to several chemokines (30). On the other
hand, Sozzani et al. (31) have previously reported that IL-10
enhanced the chemotactic migratory capacity of human monocytes. To
assess the relationship between activation and maturation states of DCs
and their migratory property, the effect of dual stimulations of
TNF- and IL-10 on the ability of monocyte-derived DCs to migrate in
response to RANTES was examined using a Transwell cell culture chamber.
Fig. 3
B shows that monocyte-derived DCs following
stimulation with TNF- exhibited lower chemotactic migratory
capacity, whereas IL-10 enhanced the capacity of these cells to migrate
as compared with that of unstimulated cells. On the other hand,
treatment of immature monocyte-derived DCs with the combination of
TNF- and IL-10 exhibited similar migratory capacity to that of
unstimulated cells.
We further evaluated the allogeneic T-cell stimulatory capacity of
monocyte-derived DCs following dual stimulations with TNF- and IL-10
(Fig. 3C). DCs stimulated with TNF- possessed a high
allostimulatory capacity as compared with untreated DCs whereas IL-10
suppressed the ability of DCs to stimulate allogeneic T-cell
proliferation. On the other hand, dual stimulations of the cells
with TNF- and IL-10 inhibited their respective regulatory
effects. These stimulatory effect correlated with cell surface
expression levels of CD86 and HLA-DR. These results indicate that IL-10
represses TNF--induced functional changes of monocyte-derived DCs.
Repression of TNF--induced activation of ERK2, SAPK/JNK or
p38mapk by IL-10 in monocyte-derived DCs
Engagement of TNF- or IL-10 by their respective receptors
increases the tyrosine phosphorylation of targeted intracellular
proteins in various cell types, and these intracellular events appear
to be crucial for some functions of these cells (18, 20, 21, 22, 23, 24, 25, 26). However,
much less is known about signaling events associated with protein
tyrosine phosphorylation cascades in DCs. Therefore, we examined the
potential involvement of TNF- or IL-10-induced tyrosine
phosphorylation events in functionality (Fig. 4A). Under the starved condition, several
intracellular proteins were weakly tyrosine phosphorylated in
unstimulated DCs whereas elevated tyrosine phosphorylation appeared in
various proteins in DCs stimulated with TNF-. Stimulation of cells
with IL-10 caused tyrosine phosphorylation of several different
intracellular proteins. On the other hand, dual stimulations with
TNF- and IL-10 initiated distinct tyrosine phosphorylation events,
and the degree of tyrosine phosphorylation of several target proteins
were significantly lower than these events following TNF-
stimulation. To address direct involvement of IL-10 in down-regulation
of TNF--induced tyrosine phosphorylation of intracellular proteins,
the cells were unstimulated or stimulated with TNF- and IL-10 in the
presence of a mAb to IL-10. Treatment of the cells with IL-10 and
anti-IL-10 mAb suppressed IL-10-induced protein tyrosine
phosphorylation events, whereas anti-IL-10 mAb inhibited
IL-10-mediated repression of TNF--induced tyrosine phosphorylation
of intracellular proteins. These results indicate that dual
stimulations with TNF- and IL-10 antagonize their respective several
tyrosine phosphorylation events in monocyte-derived DCs.
|
initiates activation of various members of the MAPK families including
ERK2, SAPK/JNK, and p38mapk (23, 24, 25, 26),
whereas stimulation with IL-10 does not activate the
p21ras/Raf/ERK pathway in several cells and
cell lines (20, 27). To clarify the potential involvement of ERK2,
SAPK/JNK, and p38mapk in the changes of DC
properties, cells were unstimulated or stimulated with TNF-, IL-10,
or the combination of these stimuli, and the level of MAPKs
phosphorylations were assessed by immunoblotting with respective
anti-tyrosine phosphorylated MAPKs mAbs (Fig. 4, B-D).
Stimulation of the cells with TNF- increased the amounts of tyrosine
phosphorylated ERK2, SAPK/JNK, and p38mapk,
whereas IL-10 failed to induce tyrosine phosphorylation of these MAPKs.
On the other hand, dual stimulations with TNF- and IL-10 resulted in
significantly reduced tyrosine phosphorylation of these MAPKs when
compared with TNF- stimulation. The total amounts of these MAPKs
were unchanged following stimulation.
Enzymatically activated ERK2, SAPK/JNK, or
p38mapk phosphorylate their transcription
factors substrates (ElK-1, c-Jun, or ATF-2, respectively) in response
to a variety of cellular stimuli, and these events result in regulation
of numerous immune/inflammatory response gene expressions (18, 23, 24, 25, 26).
Therefore, we examined whether any kinase activities were associated
with changes in the amount the tyrosine phosphorylation forms of these
MAPKs (Fig. 4, B-D). Indeed, the kinase assays clearly show
that TNF- triggered activation of ERK2, SAPK/JNK, and
p38mapk, whereas IL-10 failed to activate
them. Conversely, dual stimulations using TNF- and IL-10 resulted in
reduction of TNF--induced kinase activities of MAPKs. These results
were concomitant with the amount of the tyrosine phosphorylation form
of these MAPKs. These results indicate that ERK2, SAPK/JNK, and
p38mapk are targets for IL-10-mediated
repression of TNF--induced activation of PTK-dependent signaling
events in monocyte-derived DCs.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-induced morphological, phenotypical, and functional changes are
selectively modulated by IL-10 in monocyte-derived DCs. Furthermore, we
show that IL-10 represses TNF--induced signaling events involving
ERK2, SAPK/JNK, and p38mapk in these cells.
We demonstrated that IL-10 inhibited TNF--induced up-regulation of
the cell surface expressions of CD86 and HLA-DR, but not CD83 (Fig. 1),
whereas IL-10 failed to abolish TNF--induced maturation-associated
homotypic aggregation of monocyte-derived DCs (Fig. 2). On the other
hand, dual stimulations of TNF- and IL-10 antagonized their
respective effects on the capacities for endocytosis, chemotactic
migration, and allogeneic T cell stimulation (Fig. 3). These results
suggest that IL-10 may selectively modulate TNF--induced changes of
the properties of monocyte-derived DCs. TNF- produced by activated
monocytes/macrophages plays a crucial role in the promotion of DC
development and their properties (14, 17), and IL-10 derived from
monocytes, as well as activated Th cells, negatively regulate several
DC properties (15, 16). Furthermore, it has been shown that DCs possess
the ability to produce TNF- and IL-10 (14, 15, 16). Thus, our results
imply that TNF- and IL-10 reciprocally control activation and
maturation states of DCs in vivo.
Receptor engagement with TNF- or IL-10 initiates intracellular
events in various cell types (10, 18, 23, 24, 25, 26). To the best of our
knowledge, we are the first to have detected PTK-dependent
intracellular signaling events in DCs (Fig. 4A). We observed
that treatment of monocyte-derived DCs with the PTK inhibitors,
genestein or herbimycin A, suppressed TNF-- or IL-10-induced changes
in several DC properties (data not shown). Furthermore, we demonstrated
that stimulation of monocyte-derived DCs with either TNF- or IL-10
induced distinct tyrosine phosphorylation of intracellular proteins in
these cells (Fig. 4A). These results suggest that
PTK-dependent cascades may be involved in their respective effects on
the activation and maturation states of monocyte-derived DCs.
Previous studies have shown that TNF--induced tyrosine
phosphorylation of ERK2, SAPK/JNK, and
p38mapk resulted in their enzymatic
activation leading to structural and functional changes in various
cell type (23, 24, 25, 26). We demonstrated that stimulation of
monocyte-derived DCs with TNF- induced tyrosine phosphorylation
and activation of ERK2, SAPK/JNK, and
p38mapk, whereas IL-10 failed to induce
these events (Fig. 4, B-D). These results imply that ERK2,
SAPK/JNK, and p38mapk may be involved in
TNF--induced changes of DC properties, whereas these MAPKs may not
be responsible for IL-10-induced regulation of these cells.
Previous studies have shown that IL-10 causes activation of JAK1, Tyk2,
PI-3 kinase, and p70 S6 kinase (21, 22). However, these intracellular
events may not be involved in the anti-inflammatory properties of
IL-10 (21, 22). Although the signaling events for IL-10-induced
inhibitory effects are unknown, PTK-dependent cascades probably play a
role. Indeed, we showed that dual stimulations with TNF- and IL-10
abolished TNF--induced tyrosine phosphorylation-dependent kinase
activities of ERK2, SAPK/JNK, and p38mapk
(Fig. 4, B-D). Recent studies have shown that the blockage
in the p21ras/Raf/ERK2 and SAPK/JNK
pathways leads to anergic states in T cells and monocytes (27, 32, 33).
These results led us to hypothesize that the IL-10-mediated signaling
may repress TNF--induced activation of ERK2, SAPK/JNK, and
p38mapk. Thus, the blockage in the MAPKs
cascades may lead to the suppressive effect of IL-10 on TNF--induced
changes of monocyte-derived DCs. Conversely, the potential effect of
TNF- on IL-10-mediated signaling remains unclear. We showed that
TNF- inhibited IL-10-induced phenotypic and functional changes
(Figs. 1 and 3). Previous studies have shown that TNF- also
activates a family of JAK/STAT, PI-3 kinase, and p70 S6 kinase in
several cells (34, 35, 36), suggesting that these molecules may not be
involved in the suppressive effect of TNF- on IL-10-mediated changes
of the properties of monocyte-derived DCs. Further study is needed to
determine the molecular mechanism underlying these phenomena.
The molecular mechanism by which IL-10 blocks PTK-mediated activation of ERK2, SAPK/JNK, and p38mapk cascades remain unclear. We (37) and others (38, 39, 40) have previously suggested that a family of protein tyrosine phosphatases may exist to antagonize a large number of kinases, and these phosphatases may be involved in dominant negative signaling in certain cells. Furthermore, a series of recent studies have shown that the phosphatases specifically dephosphorylate several members of the MAPK families (41). Although the precise relationship between IL-10-mediated intracellular events and their respective phosphatases remain unknown, our data suggests that the downstream section of the IL-10-mediated signaling cascade may negatively regulate PTK-dependent cascades involving MAPKs.
In summary, our results suggest that PTK-dependent cascades may be
involved in TNF-- or IL-10-mediated regulation of monocyte-derived
DCs. Furthermore, the blockage in the MAPKs cascades may contribute to
suppressive effects of IL-10 on TNF--induced changes of DC
properties. Further characterization of the molecular events of DCs may
elucidate the regulation of DC properties by extracellular stimuli.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Abbreviations used in this paper: DCs, dendritic cells; ECL, enhanced chemiluminescence; ERK, extracellular signal-regulated kinase; FITC-DX, FITC labeled-dextran; HRP, horseradish peroxidase; JAK, Janus kinase; LY, lucifer yellow; MAPK, mitogen-activated protein kinase; PI-3 kinase, phosphatidylinositol 3-kinase; PE, phycoerythin; PTK, protein tyrosine kinase; SAPK/JNK, stress-activated protein kinase/c-jun N-terminal kinase; PVDF, polyvinylidene difluoride; ATF, activating transcription factor.
Received for publication October 13, 1998. Accepted for publication December 23, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
cooperate in the generation of dendritic Langerhans cells. Nature 360:258.[Medline]
. J. Exp. Med. 179:1109.B activation prevents cell death. Cell 87:565.[Medline]
. Proc. Natl. Acad. Sci. USA 94:13169.-induced activation of c-jun N-terminal kinase is mediated by TRAF-2. EMBO J. 16:1080.[Medline]
B and c-jun N-terminal kinase (JNK/SAPK) pathways at TNF receptor- associated factor 2. Proc. Natl. Acad. Sci. USA 94:9792. in mouse bone marrow-derived macrophages. J. Immunol. 159:4491.[Abstract]
on insulin signaling and glucose uptake in 3T3–L1 adipocytes. Diabetes 47:24.[Abstract]
RIIB1 activates Src homology 2-containing protein tyrosine phosphatase-1. J. Immunol. 161:2716.This article has been cited by other articles:
|
|
 |
|
  L. P. Carvalho, E. J. Pearce, and P. Scott Functional Dichotomy of Dendritic Cells following Interaction with Leishmania braziliensis: Infected Cells Produce High Levels of TNF-{alpha}, whereas Bystander Dendritic Cells Are Activated to Promote T Cell Responses J. Immunol., November 1, 2008; 181(9): 6473 - 6480. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Escors, L. Lopes, R. Lin, J. Hiscott, S. Akira, R. J. Davis, and M. K. Collins Targeting dendritic cell signaling to regulate the response to immunization Blood, March 15, 2008; 111(6): 3050 - 3061. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Izadi, A. T. Motameni, T. C. Bates, E. R. Olivera, V. Villar-Suarez, I. Joshi, R. Garg, B. A. Osborne, R. J. Davis, M. Rincon, et al. c-Jun N-Terminal Kinase 1 Is Required for Toll-Like Receptor 1 Gene Expression in Macrophages Infect. Immun., October 1, 2007; 75(10): 5027 - 5034. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Iijima, Y. Yanagawa, J. M. Clingan, and K. Onoe CCR7-mediated c-Jun N-terminal kinase activation regulates cell migration in mature dendritic cells Int. Immunol., September 1, 2005; 17(9): 1201 - 1212. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y.-L. Lin, Y.-C. Liang, S.-S. Lee, and B.-L. Chiang Polysaccharide purified from Ganoderma lucidum induced activation and maturation of human monocyte-derived dendritic cells by the NF-{kappa}B and p38 mitogen-activated protein kinase pathways J. Leukoc. Biol., August 1, 2005; 78(2): 533 - 543. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Wilflingseder, B. Mullauer, H. Schramek, Z. Banki, M. Pruenster, M. P. Dierich, and H. Stoiber HIV-1-Induced Migration of Monocyte-Derived Dendritic Cells Is Associated with Differential Activation of MAPK Pathways J. Immunol., December 15, 2004; 173(12): 7497 - 7505. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Gerlini, A. Tun-Kyi, C. Dudli, G. Burg, N. Pimpinelli, and F. O. Nestle Metastatic Melanoma Secreted IL-10 Down-Regulates CD1 Molecules on Dendritic Cells in Metastatic Tumor Lesions Am. J. Pathol., December 1, 2004; 165(6): 1853 - 1863. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Nakahara, H. Uchi, K. Urabe, Q. Chen, M. Furue, and Y. Moroi Role of c-Jun N-terminal kinase on lipopolysaccharide induced maturation of human monocyte-derived dendritic cells Int. Immunol., December 1, 2004; 16(12): 1701 - 1709. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D Lio, G Candore, A Crivello, L Scola, G Colonna-Romano, L Cavallone, E Hoffmann, M Caruso, F Licastro, C M Caldarera, et al. Opposite effects of interleukin 10 common gene polymorphisms in cardiovascular diseases and in successful ageing: genetic background of male centenarians is protective against coronary heart disease J. Med. Genet., October 1, 2004; 41(10): 790 - 794. [Full Text] [PDF]
|
|
|
|
|
|
P. Anderson, K. Phillips, G. Stoecklin, and N. Kedersha Post-transcriptional regulation of proinflammatory proteins J. Leukoc. Biol., July 1, 2004; 76(1): 42 - 47. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Messmer, H. Yang, G. Telusma, F. Knoll, J. Li, B. Messmer, K. J. Tracey, and N. Chiorazzi High Mobility Group Box Protein 1: An Endogenous Signal for Dendritic Cell Maturation and Th1 Polarization J. Immunol., July 1, 2004; 173(1): 307 - 313. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Chen, J. Guo, M. Yang, C. Han, M. Zhang, W. Chen, Q. Liu, J. Wang, and X. Cao Cyclosporin A impairs dendritic cell migration by regulating chemokine receptor expression and inhibiting cyclooxygenase-2 expression Blood, January 15, 2004; 103(2): 413 - 421. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Xie, J. Qian, S. Wang, M. E. Freeman III, J. Epstein, and Q. Yi Novel and Detrimental Effects of Lipopolysaccharide on In Vitro Generation of Immature Dendritic Cells: Involvement of Mitogen-Activated Protein Kinase p38 J. Immunol., November 1, 2003; 171(9): 4792 - 4800. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Sato, N. Yamashita, M. Baba, and T. Matsuyama Modified myeloid dendritic cells act as regulatory dendritic cells to induce anergic and regulatory T cells Blood, May 1, 2003; 101(9): 3581 - 3589. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Ding, D. Chen, A. Tarcsafalvi, R. Su, L. Qin, and J. S. Bromberg Suppressor of Cytokine Signaling 1 Inhibits IL-10-Mediated Immune Responses J. Immunol., February 1, 2003; 170(3): 1383 - 1391. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. G. Brinker, H. Garner, and J. R. Wright Surfactant protein A modulates the differentiation of murine bone marrow-derived dendritic cells Am J Physiol Lung Cell Mol Physiol, January 1, 2003; 284(1): L232 - L241. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Lejeune, L. Dumoutier, S. Constantinescu, W. Kruijer, J. J. Schuringa, and J.-C. Renauld Interleukin-22 (IL-22) Activates the JAK/STAT, ERK, JNK, and p38 MAP Kinase Pathways in a Rat Hepatoma Cell Line. PATHWAYS THAT ARE SHARED WITH AND DISTINCT FROM IL-10 J. Biol. Chem., September 6, 2002; 277(37): 33676 - 33682. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Steinbrink, E. Graulich, S. Kubsch, J. Knop, and A. H. Enk CD4+ and CD8+ anergic T cells induced by interleukin-10-treated human dendritic cells display antigen-specific suppressor activity Blood, April 1, 2002; 99(7): 2468 - 2476. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Yanagawa, N. Iijima, K. Iwabuchi, and K. Onoe Activation of extracellular signal-related kinase by TNF-{alpha} controls the maturation and function of murine dendritic cells J. Leukoc. Biol., January 1, 2002; 71(1): 125 - 132. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Kelly, A. Lynch, E. Vereker, Y. Nolan, P. Queenan, E. Whittaker, L. A. J. O'Neill, and M. A. Lynch The Anti-inflammatory Cytokine, Interleukin (IL)-10, Blocks the Inhibitory Effect of IL-1beta on Long Term Potentiation. A ROLE FOR JNK J. Biol. Chem., November 30, 2001; 276(49): 45564 - 45572. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S.-i. Hashimoto, I. Komuro, M. Yamada, and K. S. Akagawa IL-10 Inhibits Granulocyte-Macrophage Colony-Stimulating Factor-Dependent Human Monocyte Survival at the Early Stage of the Culture and Inhibits the Generation of Macrophages J. Immunol., October 1, 2001; 167(7): 3619 - 3625. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Puig-Kroger, M. Relloso, O. Fernandez-Capetillo, A. Zubiaga, A. Silva, C. Bernabeu, and A. L. Corbi Extracellular signal-regulated protein kinase signaling pathway negatively regulates the phenotypic and functional maturation of monocyte-derived human dendritic cells Blood, October 1, 2001; 98(7): 2175 - 2182. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Ameixa and J. S. Friedland Down-Regulation of Interleukin-8 Secretion from Mycobacterium tuberculosis-Infected Monocytes by Interleukin-4 and -10 but Not by Interleukin-13 Infect. Immun., April 1, 2001; 69(4): 2470 - 2476. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-F. Arrighi, M. Rebsamen, F. Rousset, V. Kindler, and C. Hauser A Critical Role for p38 Mitogen-Activated Protein Kinase in the Maturation of Human Blood-Derived Dendritic Cells Induced by Lipopolysaccharide, TNF-{{alpha}}, and Contact Sensitizers J. Immunol., March 15, 2001; 166(6): 3837 - 3845. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. G. A. Delemarre, P. G. Hoogeveen, M. de Haan-Meulman, P. J. Simons, and H. A. Drexhage Homotypic cluster formation of dendritic cells, a close correlate of their state of maturation. Defects in the biobreeding diabetes-prone rat J. Leukoc. Biol., March 1, 2001; 69(3): 373 - 380. [Abstract] [Full Text]
|
|
|
|
|
|
K. Sato, H. Kawasaki, H. Nagayama, M. Enomoto, C. Morimoto, K. Tadokoro, T. Juji, and T. A. Takahashi Signaling events following chemokine receptor ligation in human dendritic cells at different developmental stages Int. Immunol., February 1, 2001; 13(2): 167 - 179. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Puig-Kroger, F. Sanz-Rodriguez, N. Longo, P. Sanchez-Mateos, L. Botella, J. Teixido, C. Bernabeu, and A. L. Corbi Maturation-Dependent Expression and Function of the CD49d Integrin on Monocyte-Derived Human Dendritic Cells J. Immunol., October 15, 2000; 165(8): 4338 - 4345. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Yayoshi-Yamamoto, I. Taniuchi, and T. Watanabe FRL, a Novel Formin-Related Protein, Binds to Rac and Regulates Cell Motility and Survival of Macrophages Mol. Cell. Biol., September 15, 2000; 20(18): 6872 - 6881. [Abstract] [Full Text]
|
|
|
|
|
|
K. M. Ardeshna, A. R. Pizzey, S. Devereux, and A. Khwaja The PI3 kinase, p38 SAP kinase, and NF-kappa B signal transduction pathways are involved in the survival and maturation of lipopolysaccharide-stimulated human monocyte-derived dendritic cells Blood, August 1, 2000; 96(3): 1039 - 1046. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Nagayama, K. Sato, H. Kawasaki, M. Enomoto, C. Morimoto, K. Tadokoro, T. Juji, S. Asano, and T. A. Takahashi IL-12 Responsiveness and Expression of IL-12 Receptor in Human Peripheral Blood Monocyte-Derived Dendritic Cells J. Immunol., July 1, 2000; 165(1): 59 - 66. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Sato, H. Kawasaki, H. Nagayama, M. Enomoto, C. Morimoto, K. Tadokoro, T. Juji, and T. A. Takahashi TGF-{beta}1 Reciprocally Controls Chemotaxis of Human Peripheral Blood Monocyte-Derived Dendritic Cells Via Chemokine Receptors J. Immunol., March 1, 2000; 164(5): 2285 - 2295. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Tan, T. Town, M. Saxe, D. Paris, Y. Wu, and M. Mullan Ligation of Microglial CD40 Results in p44/42 Mitogen-Activated Protein Kinase-Dependent TNF-{alpha} Production That Is Opposed by TGF-{beta}1 and IL-10 J. Immunol., December 15, 1999; 163(12): 6614 - 6621. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. A. Terkeltaub IL-10: An "Immunologic Scalpel" for Atherosclerosis? Arterioscler Thromb Vasc Biol, December 1, 1999; 19(12): 2823 - 2825. [Full Text] [PDF]
|
|
|
|
|
|
R. LaMonica, S. S. Kocer, J. Nazarova, W. Dowling, E. Geimonen, R. D. Shaw, and E. R. Mackow VP4 Differentially Regulates TRAF2 Signaling, Disengaging JNK Activation while Directing NF-kappa B to Effect Rotavirus-specific Cellular Responses J. Biol. Chem., June 1, 2001; 276(23): 19889 - 19896. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. C. Urban, N. Willcox, and D. J. Roberts A role for CD36 in the regulation of dendritic cell function PNAS, July 17, 2001; 98(15): 8750 - 8755. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
