| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
controls the maturation and function of murine dendritic cells
Division of Immunobiology, Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan
Correspondence: Dr. K. Onoé, Division of Immunobiology, Institute for Genetic Medicine, Hokkaido University, Kita-15, Nishi-7, Kita-ku, Sapporo, 060-0815 Japan. E-mail: kazunori{at}imm.hokudai.ac.jp
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-induced maturation of murine spleen-derived DC. TNF-
increased surface expressions of major histocompatibility
(MHC) and costimulatory molecules on DC in a dose-dependent
manner. High (40 ng/ml) and low (0.4 ng/ml) concentrations of TNF-
markedly enhanced ERK1/2 activation in DC, and this activation was
blocked completely by PD98059, a selective inhibitor of the ERK
pathway. When DC were treated with TNF- at a low but not a high
concentration, PD98059 notably enhanced surface expressions of the MHC
and costimulatory molecules and allostimulatory capability of the DC.
Interleukin (IL)-12 production was enhanced significantly by PD98059 in
DC treated with low or high concentration of TNF-. These findings
suggest that TNF--induced ERK activation negatively controls
maturation and IL-12 production in murine DC.
Key Words: MAPK • PD98059 • IL-12 • APC
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Signal transduction via mitogen-activated protein kinases (MAPK) plays
an important role in cellular responses including growth factor-induced
cell proliferation, differentiation, and survival. Three groups of MAPK
have been identified in mammals: the extracellular signal-related
kinase (ERK) subfamily, p44mapk/erk1 (ERK1) and p42mapk/erk2 (ERK2)
[5
, 6
]; the stress-activated protein
kinase/c-Jun N-terminal kinase (SAPK/JNK) subfamily, p46 SAPK/JNK and
p54 SAPK/JNK isoforms and their variants [7
,
8
]; and the p38mapk subfamily [9
,
10
]. MAPK are activated following engagement of a variety
of cell-surface receptors via dual tyrosine and threonine
phosphorylation [11
12
13
14
]. This phosphorylation takes
place at upstream MAPK kinases. The activated MAPK subsequently
phosphorylate their respective substrates on serine or threonine
residues. Previous studies have shown that ligation of tumor necrosis
factor (TNF)- with TNF-R1 initiates activation of ERK, SAPK/JNK, and
p38mapk in several cells and cell lines [11
12
13
14
].
TNF-, which is highly expressed in inflammatory sites, promotes DC
maturation [15
16
17
]. The maturation of DC is inhibited
by interleukin (IL)-10, an anti-inflammatory cytokine
[18
, 19
]. Recently, it was shown that
TNF- induced tyrosine phosphorylation and activation of ERK,
p38mapk, and SAPK/JNK in human monocyte-derived DC, and phosphorylation
of these MAPK was suppressed by IL-10 [20
]. Thus, it
seems that these MAPK are involved in the signal transduction for DC
maturation. However, the precise role of respective MAPK in the DC
maturation is not fully understood.
Several groups have succeeded in generating large numbers of functional DC and Langerhans cells (LC) in the murine and the human systems by treating the DC precursors with granulocyte-macrophage colony-stimulating factor (GM-CSF) alone or in combination with other growth factors [21 ]. However, such DC could be maintained only for limited time periods, up to 3 months. Although it was described that growth factor-dependent, long-term DC lines were established from mouse fetal or newborn skin [22 , 23 ], these lines remained in an immature phenotype and were unable to be induced to mature type in vitro.
Recently, Winzler and colleagues [17
] established a
growth factor-dependent, immature DC line from splenocytes of adult
C57BL/6 mice, which could proliferate continuously for more than one
year. Although this DC line showed an immature phenotype, various
activating signals, such as living bacteria or cytokines including
TNF- and IL-1, promoted full maturation of this precursor. Indeed,
during this culture process, expressions of MHC and costimulatory
molecules, CD80, CD86, and CD40, were up-regulated on their surface.
Thus, this in vitro differentiation system appears to be useful to
study precisely the signal transduction system involved in DC
maturation.
In the present study, using the method of Winzler and colleagues
[17
], we established a growth factor-dependent, immature
DC line from splenocytes of BALB/c mice and analyzed the signal
transduction system involved in DC maturation and function. We
demonstrate herein that the ERK pathway is responsible for the
inhibition of TNF--induced DC maturation and function.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Culture media
Culture media were Iscove’s modified Dulbecco’s medium (IMDM)
and RPMI-1640 (Sigma Chemical Co., St. Louis, MO) supplemented with 100
IU/ml penicillin, 100 µg/ml streptomycin, 600 µg/ml
L-glutamine, and 50 µM 2-mercaptoethanol (2-ME).
Fibroblast supernatants (SN) from NIH/3T3 cells were collected from
confluent cultures with IMDM containing 10% heat-inactivated fetal
calf serum (FCS).
Reagents and antibodies (Abs)
Recombinant murine GM-CSF and TNF- were purchased from
PeproTech (London, UK). PD98059 was obtained from Calbiochem (La Jolla,
CA) and used at 50 µM, because it has been shwon that this
concentration specifically inhibits phosphorylation of ERK1/2
[24
]. Fluorescein isothiocyanate (FITC)-conjugated
anti-mouse CD11c monoclonal antibody (mAb; HL3), FITC-conjugated
anti-mouse CD11b mAb (M1/7), FITC-conjugated anti-mouse B220 mAb
(RA3-6B2), FITC-conjugated anti-mouse CD1d mAb (1B1), FITC-conjugated
anti-mouse CD4 mAb (L3T4), FITC-conjugated anti-mouse CD40 mAb (3/23),
phycoerythrin (PE)-conjugated anti-mouse CD80 mAb (16-10A1),
PE-conjugated anti-mouse CD86 mAb (GL1), PE-conjugated anti-mouse CD44
mAb (KM114), PE-conjugated anti-mouse CD8 mAb (53-6.7),
biotin-conjugated anti-mouse CD49d mAb (MFR4.B), biotin-conjugated
anti-mouse intercellular adhesion molecule-1 (ICAM-1) mAb (3E2),
biotin-conjugated anti-mouse H-2Kd mAb (SF1-1.1), and
streptavidin Cy-ChromeTM were obtained from PharMingen (La Jolla, CA).
As control immunoglobulin G (IgG), FITC-conjugated hamster IgG,
FITC-conjugated rat IgG1, biotin-conjugated rat IgG2a, and
PE-conjugated rat IgG1 were obtained from PharMingen. FITC-conjugated
rat IgG2b, PE-conjugated rat IgG2a, and biotin-conjugated mouse IgG2b
were purchased from Immunotech (Marseille, France). Biotin-conjugated
mouse IgG2a were purchased from DAKO (Copenhagen, Denmark).
PE-conjugated hamster IgG was obtained from Caltag Laboratories
(Burlingame, CA).
Generation of DC
Growth factor-dependent cultures of immature DC from murine
splenocytes were performed as described in previous studies
[17
, 25
]. Spleens were removed from BALB/c
mice, and single-cell suspensions were prepared by passing through a
stainless mesh. Erythrocytes were lysed by treatment with ammonium
chloride. Remaining unfractionated cell populations were plated at a
density of 5 x 105 cells/ml. Culture medium for
generation and expansion of DC was IMDM containing 10% FCS, 30%
NIH/3T3 SN, and 5 ng/ml mouse recombinant GM-CSF (henceforth referred
to as R1 medium). Cultures were fed with fresh R1 medium every 3–4
days. First passages of DC-enriched cultures were performed after 2
weeks. Suspended and weakly adherent cells were propagated. Clusters of
adherent cells with a dendritic morphology were detached with 3 mM
ethylenediaminetetraacetic acid (EDTA) and collected. After 2 months of
culture, homogeneous growth factor-dependent DC were generated.
Experiments were performed with the cells continuously cultured for
more than 4 months. This growth factor-dependent DC population is
referred to as BC1 cells.
In vitro kinase assays
BC1 cells (2x106) in 2 ml IMDM containing 5% FCS
were stimulated with TNF- for the indicated times at 37°C. For
inhibiting the ERK pathway, the cells were pretreated with 50µM
MAPK/ERK kinase (MEK) inhibitor, PD98059, for 1 h at 37°C before
being stimulated with TNF-. Reactions were stopped by rapidly
cooling on ice. The cells were washed by ice-cold phosphate-buffered
saline (PBS), and then in vitro kinase assays for ERK were performed
using a commercially available p44/42 MAPK assay kit (New England
BioLabs, Beverly, MA) according to the manufacturer’s instructions.
The cells were lysed with 250 µl lysis buffer [1% Triton X-100, 20
mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM
ethyleneglycol-bis(ß-aminoethyl
ether)-N,N,N',N'-tetraacetic acid (EGTA), 2.5 mM sodium
pyrophosphate, 1 mM ß-glycerol phosphate, 1 mM sodium orthovanadate,
1 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride], and the
lysates were subjected to immunoprecipitation with immobilized
antiphospho-ERK1/2 mAb. The complexes were washed twice with lysis
buffer and twice with kinase buffer [25 mM Tris-HCl (pH 7.5), 5 mM
ß-glycerol phosphate, 2 mM dithiothreitol, 0.1 mM sodium
orthovanadate, and 10 mM MgCl2], before resuspending in 50
µl kinase buffer containing 100 µM adenosine triphosphate and 1
µg Elk-1. Reactions were terminated with addition of 25 µl 3x
sodium dodecyl sulfate (SDS) sample buffer [187.5 mM Tris-HCl (pH
6.8), 6% SDS, 30% glycerol, 150 mM dithiothreitol, 0.3% bromphenol
blue]. Samples were heated at 95°C for 5 min, separated by 15%
SDS-polyacrylamide gel electrophoresis (PAGE), and transferred onto a
polyvinylidene-difluoride membrane. The membrane was probed with
antiphospho-Elk-1 mAb and developed with horseradish peroxidase
(HRP)-conjugated secondary Ab by enhanced chemiluminescence.
Flow cytometry
BC1 cells were pretreated with or without 50 µM PD98059 for
1 h and then treated with 0.4 or 40 ng/ml TNF- for 24 h in
the presence or absence of PD98059 in R1 medium. The cells were
detached with 3 mM EDTA (3 min at 37°C). These cells were incubated
with 2.4G2 (rat anti-mouse FcII/III receptor, CD32) SN to prevent
binding to FcRII/III and then stained using FITC- , PE- , or
biotin-conjugated mAb and streptavidin-Cy-ChromeTM [26
].
Flow cytometry was performed on EPICS® XL (Coulter Co., Miami, FL),
as described in a previous study [27
].
Assessment of apoptosis by flow cytometry
Apoptosis was assessed using fluorescent-labeled annexin-V,
Annexin-V-FLUOS© (Roche Diagnostics GmbH, Mannheim,
Germany), according to the manufacturer’s instructions. BC1 cells were
pretreated with or without 50 µM PD98059 for 1 h and then with
0.4 or 40 ng/ml TNF- in the presence or absence of PD98059 for
24 h in R1 medium. Cells were detached and double-stained with
Annexin-V-FLUOS© and propidium iodide (PI) and analyzed by
flow cytometry.
Mixed leukocyte reaction (MLR) assay
Allogeneic MLR was performed using nylon nonadherent splenic
lymphocytes from C57BL/6 mice as responder cells, as described in a
previous study [28
]. BC1 cells were pretreated with or
without 50 µM PD98059 for 1 h and then with 0.4 or 40 ng/ml
TNF- for 24 h in the presence or absence of PD98059 in R1
medium. Cells were detached and washed three times with RPMI-1640
containing 10% FCS, 100 IU/ml penicillin, 100 µg/ml streptomycin,
600 µg/ml L-glutamine, and 50 µM 2-ME (complete
RPMI-1640). BC1 cells were irradiated (30 Gy) and cocultured with
responder cells (4x105 cells) in 200 µl complete
RPMI-1640 at 37°C in 5% CO2. After 68 h, cultures
were pulsed with 0.5 µCi/well [3H]thymidine (Amersham,
Tokyo, Japan) for 4 h and then harvested onto glass fiber.
Incorporation of [3H]thymidine was measured with a liquid
scintillation counter (MicroBeta Plus; Wallac, Turku, Finland).
Measurement of IL-12 p70 by enzyme-linked immunosorbent assay
(ELISA)
BC1 cells were pretreated with or without 50 µM PD98059 for
1 h and then with 0.4 or 40 ng/ml TNF- for 24 h in the
presence or absence of PD98059 at a density of 4 x
105 cells/ml. Culture supernatants were subjected to
quantification of the protein level of IL-12p70 by ELISA using a
commercially available mouse IL-12p70 immunoassay kit (BioSource
International, Camarillo CA) according to the manufacturer’s
instructions.
Statistical analysis
The Student’s t-test was used to analyze data for
significant differences. P values <0.05 were regarded as
significant.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
(lymphoid) molecules,
respectively. BC1 cells expressed CD11b but not CD8. The majority of
BC1 cells expressed moderate levels of MHC class I and class II
molecules (H2-Kint and I-Aint), but a small
proportion of these cells expressed high levels of class I and class II
molecules (H2-Kbright and I-Abright). The same
patterns were observed when CD80 and CD86 expressions were analyzed.
Double-color flow cytometric analysis of unstimulated cells showed that
I-Abright cells were expressing high levels of CD86
(unpublished results). Conversely, almost no BC1 cells expressed CD40,
although a very small proportion of these cells were CD40-positive.
Considerable amounts of adhesion molecules, CD49d, ICAM-1, and CD44,
were detected on BC1 cells. It is also shown in Figure 1
that BC1 cells
are CD1d-, CD4-, and B220-negative. Altogether, these results indicate
that BC1 cells are typical, immature DC of myeloid origin.
|
(40
ng/ml) for 24 h, these cells developed larger veils and longer
dendrites and formed large floating cell aggregates
(unpublished results). We then analyzed expressions of MHC class II,
MHC class I, CD86, CD80, and CD40 on the TNF--treated BC1 cells by
flow cytometry (Fig. 2 A
). TNF- markedly up-regulated MHC class II, MHC class I, CD86,
and CD80 expressions on BC1 cells. It should be noted that almost all
BC1 cells came to express CD40 after TNF- treatment.
|
on allostimulatory capabilities
of BC1 cells, allogeneic MLR was performed using nylon-wool nonadherent
splenic lymphocytes from C57BL/6 mice as responder cells. It is shown
in Figure 2B
that TNF- (40 ng/ml) markedly enhances allostimulatory
capability of BC1 cells, compared with that of TNF- nontreated BC1
cells (Control).
Activities of ERK in TNF--treated DC
MAPK are activated by engagement of various receptors with the
ligands as well as by environmental stresses and have been shown to
mediate mitogenic and apoptotic responses [29
]. In
murine DC, it has been shown that the lipopolysaccharide (LPS) markedly
activates ERK1/2 [30
]. We then examined whether TNF-
activates ERK1/2 in BC1 cells. TNF- (0.4–40 ng/ml) increased
surface expressions of MHC class II and CD86 on BC1 cells in a
dose-dependent manner (Fig. 3 A
). Thus, 40 and 0.4 ng/ml TNF- were regarded as optimal and
suboptimal concentrations for DC maturation, respectively.
|
, and in vitro kinase assay
for ERK1/2 activation was performed using Elk-1 as a substrate for
ERK1/2. Phosphorylation of Elk-1 was determined by Western blotting
using antiphospho-Elk-1 mAb. Figure 3B
shows a slight but distinct band
of phospho-Elk-1 in unstimulated BC1 cells. Optimal and suboptimal
concentrations of TNF- markedly enhanced activation of the ERK1/2 in
BC1 cells. It has been shown that PD98059 selectively inhibits MEK, a
kinase of ERK1/2, which results in inhibition of the ERK pathway
[31
]. Indeed, PD98059 specifically inhibited the
TNF--induced ERK pathway in human monocyte-derived DC
[32
]. We then examined the effect of PD98059 on the
TNF--induced activation of ERK1/2 in BC1 cells. As shown in Figure 3B
, the activation of ERK1/2 by optimal or suboptimal concentration of
TNF- (40 or 0.4 ng/ml) was almost completely inhibited by the
treatment with PD98059.
MHC class II, CD86, and CD40 expressions and allostimulatory
capability of DC during TNF- treatment in the presence of PD98059
To elucidate the role of ERK1/2 activation by TNF- in DC
maturation, we then analyzed various surface markers including MHC and
costimulatory molecules on BC1 cells treated with or without TNF- in
the presence or absence of PD98059. Figure 4
shows that PD98059 decreases the MHC class II and CD86 expressions
on unstimulated BC1 cells but exerts no influence on the MHC class I,
CD80, and CD40 expressions. Again, optimal concentration of TNF- (40
ng/ml) markedly enhanced surface expressions of MHC class II, MHC class
I, CD86, CD80, and CD40 on BC1 cells. However, under this condition,
PD98059 showed no notable effects on the enhanced expressions of these
molecules. Conversely, in the presence of suboptimal concentration of
TNF- (0.4 ng/ml), slightly increased surface expressions of MHC
class II, MHC class I, CD86, CD80, and CD40 on BC1 cells were
considerably augmented by PD98059. Thus, ERK activation by the
suboptimal concentration of TNF- appears to be involved in the
negative regulation of DC maturation.
|
, and addition of PD98059 showed no influence on the
increased MLR. The suboptimal concentration of TNF- (0.4 ng/ml)
slightly increased allostimulation by BC1 cells. However, in this
condition, PD98059 markedly enhanced the allostimulatory capability of
BC1 cells, and this augmentation was regarded as statistically
significant when 2 x 104 or 4 x 104
BC1 cells were stimulators (P<0.05). These findings appear
to be consistent with the expression patterns of MHC and costimulatory
molecules on BC1 cells (Figs. 3
and 4)
.
|
and PD98059 on the apoptosis of BC1
cells using fluorescent-labeled annexin-V, which detects the appearance
of an early apoptotic marker, phosphatidylserine, on the cell surface
(Fig. 6
). The proportion of BC1 cells undergoing apoptosis
(annexin-V-positive and PI-negative cells) was 0.4%. Neither optimal
nor suboptimal concentration of TNF- affected apoptosis of BC1
cells. Conversely, PD98059 increased apoptosis of BC1 cells in the
control culture (P<0.01) and in the presence of a
suboptimal concentration of TNF- (P<0.05) but not in the
presence of an optimal concentration of TNF-. Nevertheless,
proportions of apoptotic BC1 cells were <2% in all the cultures
tested.
|
-induced IL-12 production of DC in the presence of PD98059-induced IL-12p70 production in BC1 cells. In the absence of
TNF-, BC1 cells produced a small amount of IL-12p70 (Fig. 7
). PD98059 showed no influence on the constitutive production of
IL-12p70. IL-12p70 production was increased significantly by treatment
with an optimal concentration of TNF- (40 ng/ml)
(P<0.01), and the increased IL-12 production was
significantly enhanced by PD98059 (P<0.01). A suboptimal
concentration of TNF- (0.4 ng/ml) alone failed to increase IL-12p70
production over the background level. However, when PD98059 was added
to the BC1 cell cultures stimulated with the suboptimal concentration
of TNF-, the IL-12p70 production was increased significantly
(P<0.01). These findings demonstrate that IL-12p70
production is negatively regulated by TNF--induced ERK activation in
murine DC.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
(Figs. 1
and 2)
. Thus far, using the similar in vitro differentiation
system of DC, a number of important findings have been described and
verified [17
, 30
, 35
36
37
]. In
the present study, we demonstrated that the ERK pathway was involved in
the regulatory signal during TNF--induced maturation and IL-12
production in BC1 cells.
Activation and maturation states of DC are regulated by various
extracellular stimuli including cytokines, costimulatory molecules, and
bacterial products. These events are accompanied with alterations of
the morphological, phenotypical, and functional properties of DC
[15
16
17
18
19
]. TNF-, an inflammatory cytokine, potently
promotes activation and maturation of DC [15
16
17
]. It
has been shown that TNF- induced tyrosine phosphorylation and
activation of ERK, p38mapk, and SAPK/JNK in human monocyte-derived DC
[20
]. However, PD98059, a specific inhibitor of the ERK
pathway, showed no notable effect on maturation of these cells
[32
]. It was also shown that LPS promoted phenotypical
maturation and ERK activation in murine DC, but again, the phenotypical
maturation was unaffected by inhibition of the ERK pathway
[30
]. Thus, the involvement of ERK activation in DC
maturation is unclear.
In the present study, we stimulated BC1 cells with various
concentrations of TNF-. Suboptimal (0.4 ng/ml) and optimal (40
ng/ml) concentrations of TNF- induced a considerable activation of
ERK in BC1 cells. We found that PD98059 showed no notable effects on
phenotypical maturation of BC1 cells induced by an optimal
concentration of TNF- (Fig. 4)
. This finding appears to be
consistent with previous studies [30
, 32
].
However, in the presence of a suboptimal concentration of TNF-,
surface expressions of MHC and costimulatory molecules were markedly
enhanced by inhibiting the ERK pathway (Fig. 4)
. In addition,
allostimulatory capability of DC treated with the suboptimal
concentration of TNF- was also enhanced by inhibiting the ERK
pathway (Fig. 5)
. Thus, the ERK activation induced by the suboptimal
concentration of TNF- appears to be involved in the negative
regulation of phenotypical and functional maturation of DC. In
unstimulated BC1 cells, a low level of ERK activation was also detected
(Fig. 3)
. Surface expressions of MHC class II and CD86 but not MHC
class I, CD80, and CD40 were reduced by inhibiting the ERK activation
in the unstimulated BC1 (Figs. 3
and 4)
. The slight but constitutive
activation of ERK may be induced by our culture condition and rather,
at least in part, be involved in sustaining the surface expressions of
MHC class II and CD86 but not MHC class I, CD80, and CD40 on these BC1
cells. The physiological role of this basal level of ERK activation
should be elucidated in future investigations.
In our culture system, BC1 cells were grown in 10% FCS IMDM
supplemented with 30% fibroblast supernatant and GM-CSF (R1 medium) as
described in a previous study [17
]. When BC1 cells were
cultured in the absence of above supplements, approximately 80% of the
cells underwent apoptosis after 24 h (unpublished results)
[30
], and a proportion of these BC1 cells were activated
(unpublished results). In this condition, the proportions of background
death and activated BC1 cells were high, and it was hard to analyze
effects of TNF- and PD98059 on the DC maturation. Thus, in the
present study, BC1 cells were driven to the mature stage by treatment
with TNF- in R1 medium, and effects of PD98059 on the
TNF--induced DC maturation were determined. In our condition, very
low proportions (<2%) of BC1 cells underwent apoptosis after
treatment of TNF- with PD98059 (Fig. 6)
, and it seemed unlikely that
the apoptotic process was substantially involved in the enhanced DC
maturation.
It has been shown that the phenotypical maturation of DC is dependent
on the activity of the nuclear factor (NF)-
B family
[3
]. Indeed, LPS and TNF- induced NF-B activation
in murine splenocyte-derived DC and human monocyte-derived DC, and
phenotypical maturations of these DC were suppressed by NF-B
inhibitors [30
, 32
]. It seems to us that
TNF- initiates activation of the signal pathway via NF-B as well
as the inhibitory signal pathway via ERK, and a balance of these signal
pathways controls the DC maturation. As shown in the present study, in
the presence of a suboptimal concentration of TNF-, the inhibitory
signal pathway via ERK might be predominant, and the inhibition of ERK
activity resulted in the promotion of DC maturation. Conversely, in the
presence of an optimal concentration of TNF-, full activation of
NF-B might occur and overcome the regulatory functions of the ERK
activation, and the full DC maturation could be seen. Under this
condition, DC maturation might be unaffected by inhibition of the ERK
pathway. In contrast, inhibition of ERK activation by PD98059 lead to a
significant enhancement of IL-12 production in BC1 cells treated with a
suboptimal or optimal concentration of TNF-. This finding
demonstrates that the inhibitory regulation by ERK activation operates
on IL-12 production even in BC1 cells that have been fully maturated by
TNF-.
Recently, it has been shown that PD98059 enhances IL-12p40 production in murine macrophages upon stimulation with LPS and bacterial DNA, and this enhanced IL-12 production correlates with IL-12p40 mRNA levels and promoter activity [38 , 39 ]. Thus, it is suggested that the ERK activation suppresses production of biologically active IL-12, which occurs primarily at the transcriptional level. Conversely, neither LPS nor bacterial DNA induced the ERK activation in murine bone marrow-derived DC, and PD98059 had no effects on the bacterial DNA-induced IL-12 production [38 ]. By contrast, it was demonstrated that LPS stimulation promoted ERK activation in murine spleen-derived DC [30 ].
In the present study, we focused on TNF--induced ERK activation in
murine spleen-derived DC and demonstrated that TNF- markedly
promoted ERK activation. The TNF--induced IL-12p70 production was
enhanced by inhibiting the ERK pathway in these DC. These findings
permit us to conclude that the ERK pathway negatively regulates IL-12
production in DC as well as in macrophages. It seems that the role of
the ERK pathway in IL-12 production is different between bone
marrow-derived DC and spleen-derived DC.
In immune responses, IL-12 plays a central role as a link between the
innate and adaptive immune systems [40
]. Thus, IL-12
induces and promotes natural killer (NK) and T cells to generate
interferon (IFN)-
and the lytic activity. In addition, IL-12
polarizes the immune system toward a primary T-helper cell (Th)-1
response. Thus, maturation and IL-12 production of DC are important
components that determine the subsequent immune responses. Elucidation
of the rather complex pathways controlling maturation and IL-12
production of DC appears to be necessary to develop the regulation
system of various immune disorders in the clinical applications.
|
ACKNOWLEDGEMENTS |
|---|
Received September 10, 2001; accepted September 13, 2001.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Proc. Natl. Acad. Sci. USA 94,13169-13174 in mouse bone marrow-derived macrophages J. Immunol. 159,4491-4497[Abstract]
-induced activation and maturation of human peripheral blood monocyte-derived dendritic cells J. Immunol. 162,3865-3872B signal transduction pathways are involved in the survival and maturation of lipopolysaccharide-stimulated human monocyte-derived dendritic cells Blood 96,1039-1046ß+ cells in zeta-associated protein 70 null mice with an accumulation of NK1.1+ CD3- NK-like cells in the thymus Blood 97,1765-1775 in the absence of IL-12 during DC-T cell cognate interaction: a new role for Fas ligand in inflammatory responses J. Exp. Med. 192,1661-1668This article has been cited by other articles:
|
|
 |
|
  Y. Yanagawa and K. Onoe Enhanced IL-10 Production by TLR4- and TLR2-Primed Dendritic Cells upon TLR Restimulation J. Immunol., May 15, 2007; 178(10): 6173 - 6180. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Q. Wang, Y. Liu, J. Wang, G. Ding, W. Zhang, G. Chen, M. Zhang, S. Zheng, and X. Cao Induction of Allospecific Tolerance by Immature Dendritic Cells Genetically Modified to Express Soluble TNF Receptor J. Immunol., August 15, 2006; 177(4): 2175 - 2185. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Kitamei, K. Iwabuchi, K. Namba, K. Yoshida, Y. Yanagawa, N. Kitaichi, M. Kitamura, S. Ohno, and K. Onoe Amelioration of experimental autoimmune uveoretinitis (EAU) with an inhibitor of nuclear factor-{kappa}B (NF-{kappa}B), pyrrolidine dithiocarbamate J. Leukoc. Biol., June 1, 2006; 79(6): 1193 - 1201. [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]
|
|
|
|
 |
|
 K. Minami, Y. Yanagawa, K. Iwabuchi, N. Shinohara, T. Harabayashi, K. Nonomura, and K. Onoe Negative feedback regulation of T helper type 1 (Th1)/Th2 cytokine balance via dendritic cell and natural killer T cell interactions Blood, September 1, 2005; 106(5): 1685 - 1693. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Valentinis, A. Bianchi, D. Zhou, A. Cipponi, F. Catalanotti, V. Russo, and C. Traversari Direct Effects of Polymyxin B on Human Dendritic Cells Maturation: THE ROLE OF I{kappa}B-{alpha}/NF-{kappa}B AND ERK1/2 PATHWAYS AND ADHESION J. Biol. Chem., April 8, 2005; 280(14): 14264 - 14271. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Do, V. L. Hegde, P. S. Nagarkatti, and M. Nagarkatti Bryostatin-1 Enhances the Maturation and Antigen-Presenting Ability of Murine and Human Dendritic Cells Cancer Res., September 15, 2004; 64(18): 6756 - 6765. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. A. Skinner, A. Reissinger, H. Shen, and M. H. Yuk Bordetella Type III Secretion and Adenylate Cyclase Toxin Synergize to Drive Dendritic Cells into a Semimature State J. Immunol., August 1, 2004; 173(3): 1934 - 1940. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. C. Reed, B. Berwin, J. P. Baker, and C. V. Nicchitta GRP94/gp96 Elicits ERK Activation in Murine Macrophages: A ROLE FOR ENDOTOXIN CONTAMINATION IN NF-{kappa}B ACTIVATION AND NITRIC OXIDE PRODUCTION J. Biol. Chem., August 22, 2003; 278(34): 31853 - 31860. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Yanagawa and K. Onoe CCR7 ligands induce rapid endocytosis in mature dendritic cells with concomitant up-regulation of Cdc42 and Rac activities Blood, June 15, 2003; 101(12): 4923 - 4929. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Yanagawa and K. Onoe CCL19 induces rapid dendritic extension of murine dendritic cells Blood, August 28, 2002; 100(6): 1948 - 1956. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
