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Departments of
*
Cell Processing,
Clinical Immunology and AIDS Research Center, and
Hematology/Oncology, Institute of Medical Science, University of Tokyo, Tokyo, Japan; and
§
Japanese Red Cross Central Blood Center, Tokyo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, and IFN-
at the transcription levels. Furthermore,
stimulation of DCs with IL-12 induced IL-12p40 transcript, but not
IL-12p35 transcript, whereas this stimulation caused the expressions of
both transcripts in Con A blasts. Stimulation of DCs with IL-12 caused
a tyrosine phosphorylation of several intracellular proteins, and the
pattern of these events were distinct from those of IL-12-stimulated
Con A blasts. IL-12 also induced tyrosine phosphorylation of IL-12Rß1
as well as recruitment of several tyrosine-phosphorylated proteins to
IL-12Rß1 in DCs and Con A blasts. Receptor engagement of DCs as well
as Con A blasts by IL-12 resulted in activation of Janus kinase 2 and
Tyk2 kinases and Stat3 and Stat4 transcription factors and the
association of these proteins to IL-12Rß1. Stimulation with IL-12
caused a tyrosine phosphorylation and enzymatic activity of a family of
mitogen-activated protein kinases, p38mapk. These results
suggest that IL-12 acts directly on DCs to induce their functional
activation via IL-12Rß1-mediated signaling events. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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). Previously, an in vitro culture
system revealed that DCs originate from CD34+
pluripotent hemopoietic progenitor cells in the bone marrow and cord
blood via myeloid lineage cells in human and murine models
(7, 8, 9, 10, 11, 12), and some DCs develop from thymic precursors via
lymphoid lineage cells in the murine system (13). Previous
studies have shown that TNF-, IL-1ß, CD40 ligand (CD40L), LPS, and
ceramide can promote DC differentiation in vitro, resulting in
irreversible morphological, phenotypical, and functional changes,
including up-regulation of MHC products and adhesion/costimulatory
molecules, down-regulation of Ag uptake, and processing capacity, which
result in enhanced TC-stimulatory capacity (13, 14, 15, 16, 17, 18).
IL-12, a heterodimeric cytokine (IL-12p70) composed of a 40-kDa subunit
(p40) and a 35-kDa subunit (p35), is produced by activated
monocytes/macrophages, DCs, neutrophils, and some B cell lines
(19, 20, 21). IL-12 play an important role for induction of
Th1 cells from naive Th0 cells, the generation of CTLs, as well as the
their production of GM-CSF, TNF-, and IFN- (19). In
contrast, IL-12 is a strong inducer of activated NK cells
(19, 20). Studies of IL-12-deficient mice revealed that
the capacity of splenocytes to produce IFN- was repressed following
bacterial, fungal, parasites, and viral infection, and these phenomenon
were involved in their defective primary immune responses
(21).
The specific effects of IL-12 on the target cell types are mediated by
the IL-12R complex, which consists of IL-12Rß1 and IL-12Rß2
(22, 23, 24, 25, 26, 27). Chua et al. have reported on the IL-12Rß1
subunit of mice (23) and humans (24), which
has strong homology to gp130, and the WSXWS motif in their
extracellular domain (23, 24). Presky et al.
(25) showed another IL-12Rß2 subunit, which also has
homology to gp130 and consists of a medium-affinity receptor with a
ß1 subunit for IL-12. A series of previous studies have shown that
IL-12Rß1 and IL-12Rß2 are mainly expressed on activated TC or NK
cells (26). Recent studies show IL-12Rß1-deficient mice
failed to produce IFN- in response to IL-12, and their splenocytes
also failed to display IL-12-induced enhancement of NK lytic activity,
suggesting that IL-12Rß1 is an essential component for the IL-12
responsiveness in vitro and in vivo (27).
The engagement of IL-12Rß1 by IL-12 induces an elaborate biochemical
program that ultimately results in the induction of a variety of
functions including cell proliferation and cytokine secretion.
Activation signals following stimulation by IL-12 involve
IL-12Rß1-dependent calcium influx and the initiation of a protein
tyrosine kinase (PTK)-dependent pathway including activation of Janus
kinase (Jak) (Jak2 and Tyk2)/Stat (Stat3 and Stat4) cascades
(28, 29, 30, 31, 32, 33, 34, 35, 36). Thierfelder et al. (31) have
previously shown that IL-12-induced IFN- production, generation of
cytolytic activity of NK cells, and induction of Th1 cells were
defective in Stat4-deficient mice.
Grohmann et al. (37, 38) have recently reported that
IL-12Rß1 and IL-12Rß2 were constitutively expressed on murine
splenic CD8+ and
CD8- DCs and their cell line at
transcriptional levels (37, 38). Furthermore, stimulation
of these DCs with IL-12 resulted in the increased secretion of
endogenous IL-12 and the enhanced class II Ag expression via nuclear
localization of NF-
B. In contrast, IL-12 reportedly induced IFN-
production in murine splenic DCs (39, 40). However,
receptor expression for IL-12 and the role of IL-12 on functional
activation of human DCs remains unclear.
In this report, we examined the expression of IL-12R in human monocyte-derived DCs and the potential roles of IL-12 in the regulation of DC properties via their downstream signaling cascades.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The medium used throughout was RPMI 1640 supplemented with 2 mM L-glutamine, 50 µg/ml streptomycin, 50 U/ml penicillin, and 10% heat-inactivated FCS. GM-CSF was kindly provided by Kirin Brewery (Tokyo, Japan). IL-4 and IL-12 were purchased from PeproTech (London, U.K.). HRP-conjugated anti-phosphotyrosine (pTyr) mAb (clone RC20) and Stat3 were purchased from Transduction Laboratories (Lexington, KY). Abs to Jak2, Tyk2, and Stat4 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The preparations of mAbs to IL-12Rß1 and the specific recognition of this mAbs were described previously (29). These mAbs were used for specific Western blotting and immunoprecipitation in the transfectants expressing IL-12Rß1 (30). p38mapk immunoblotting kits, their kinase assay kits, and HRP-conjugated secondary Abs were purchased from New England Biolabs (Beverly, MA).
In vitro generation and culture of human DCs
DCs were generated from PBMCs as described previously (9) with some modification (10, 11, 12). Briefly, PBMCs were obtained from 30 ml of leukocyte-enriched buffy coat from healthy donors by centrifugation with Ficoll-Hypaque (Pharmacia Biotech, 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 six-well plates (Costar, Cambridge, MA). After 2 h at 37°C, nonadherent cells were removed and adherent cells were collected, and these cells were negatively selected with anti-CD2 mAb-conjugated immunomagnetic beads (Dynal, Oslo, Norway) and anti-CD19 mAb-conjugated immunomagnetic beads (Dynal) to deplete CD2+ cells and CD19+ cells according to the manufacturer’s instructions. The resulting cells (>95% CD14+ cells) were used as monocytes and 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 used for subsequent experiments. These cell populations exhibited a typical DC morphology and phenotype (>95% pure as indicated by anti-CD1a mAb, anti-CD11c mAb, and anti-HLA-DR mAb staining, and < 1% CD3+cell, CD19+cell, CD14+cell, CD56+cells; Becton Dickinson, Mountain View, CA) (10, 11, 12). We also observed that CD4 was expressed in all subpopulation of monocytes-derived DCs, and there were no subpopulation of CD8+ cells (10, 11, 12).
Preparation and culture of TCs and Con A blasts
TCs were isolated from monocye-depleted cell population by E-rosetting (41), and TC preparations were typically >95% pure as indicated by anti-CD3 mAb staining (Becton Dickinson). Con A blasts were prepared from TCs by cultivation with 5 µg/ml of Con A and 10 ng/ml of IL-2 for 48 h, followed by incubation in the presence of IL-2 alone for 5 days (41). Dead cells were removed by gradient centrifugation using Histopaque 1083 (Sigma, St. Louis, MO).
Semiquantitative RT-PCR
RNA from each sample (107) was isolated
using Trizol LS reagent (Life Technologies, Gaithersburg, MD). The
first-strand cDNA kit (SuperScript Preamplication System; Life
Technologies) was used to make cDNA (20 µl) from 5 µg of each RNA.
Amplification of each cDNA (1 µl) was performed with a SuperTaq
Premix kit (Sawady Technology, Tokyo, Japan) using specific primers for
IL-12Rß1 (28) and IL-12Rß2 as follows; 5'-GTC GAC CCT
ACA ATG TGT CTG CTC TGA TTT-3' and 5'-TCA GAG CAT GAG GGA GTC ACA CCT
CAT CTT-3'. Specific primers for ß-actin and other cytokines
including GM-CSF, IL-1ß, IL-6, TNF-, IFN-, IL-12p35, and
IL-12p40 (all from Continental Laboratory Products, San Diego, CA) were
also used for amplification. Thermal cycling of IL-12Rß1 and
IL-12Rß2 primers was performed as follows: denaturation at 94°C for
1 min, anealing at 55°C for 1 min, and extention at 72°C for 1 min.
Other cytokines and ß-actin primers were anealed at 60°C, and all
cycling was performed for 35 cycles. PCR products were analyzed by
electrophoresis through 2% agarose gels and visualized under UV light
after ethidium bromide staining.
Flow cytometry
TCs, Con A blasts, monocytes, or DCs, which was treated with 0.5% mouse serum (Dako, Glostrup, Denmark) for 15 min at 4°C to block the Fc receptor, were incubated with biotin conjugated anti-IL-12Rß1 mAb for 30 min at 4°C, washed twice with cold PBS, and subsequently stained with FITC-conjugated avidin (Becton Dickinson) for 30 min at 4°C. Thereafter, the cells were washed twice and suspended in PBS containing 0.2 µg/ml propidium iodide (Sigma) to exclude dead cells. Analysis of fluorescence staining was performed with a FACSCalibur flow cytometer (Becton Dickinson) and CellQuest software.
Stimulation of Con A blasts and DCs
Con A blasts and DCs (107/5 ml) were unstimulated or stimulated with various concentrations of IL-12 (1–100 ng/ml) in the presence or absence of 1 µg/ml of control mouse IgG (cont. IgG; Sigma) or anti-IL-12Rß1 mAb for 5 min (for Western blotting and immune complex kinase assay) or 48 h (for semiquantitative RT-PCR and ELISA). The cells were washed twice with cold PBS and used for subsequent experiments.
Detection of IFN- by ELISA
The in vitro production of IFN- was assessed by ELISA as
described previously (10). The culture supernatants (5 ml)
of unstimulated or stimulated cells (107) were
collected and assayed for IFN- production. IFN- was detected in
the supernatants using a two-site sandwich ELISA (Endogen, Woburn, MA).
Samples were analyzed in serial 2-fold dilutions in duplicate; the
sensitivity of the assay was 2 pg/ml.
Western blotting, immunoprecipitation, and immune complex kinase assay
Con A blasts and DCs was starved in serum-free medium for 16 h at 37°C and subsequently kept for 4 h on ice to reduce the basal levels of tyrosine phosphorylation of intracellular proteins (12, 41). The cells (4 x 106) were untreated or stimulated with IL-12 (1–100 ng/ml) for 5 min at 37°C and washed twice in cold PBS, 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), and total cell lysates were obtained. To prepare immunoprecipitated samples, the total cell lysates (107 cells) were immunoprecipitated with an Ab to IL-12Rß1, Jak2, Tyk2, Stat3, or Stat4, and the immunocomplex was collected by protein G Sepharose 4 fast flow (Pharmacia Biotech) and washed three times with lysis buffer. The total cell lysates or the immunoprecipitates sample were suspended in 2x SDS sample buffer (313 mM Tris-HCl, pH 6.8, 10% SDS, 2% 2-ME, 50% glycerol, and 0.01% bromophenol blue) and heated for 3 min at 95°C. The samples were fractionated by 12% SDS-PAGE, transferred onto polyvinylidene difluoride PVDF membranes (Millipore, Bedford, MA), and probed with HRP-conjugated anti-pTyr mAb. Blots were visualized by enhanced chemiluminescence (ECL) (New England Biolabs). To ensure similar amounts of respective proteins in each sample, the same membrane was stripped off, reprobed with the stated Abs, and developed with HRP-conjugated secondary Abs (Santa Cruz Biotechnology) by ECL. Immunoblotting and in vitro kinase assay of p38mapk were performed with their respective kits according to the manufacturer’s instructions (12, 41).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies have shown that IL-12R is detected in TCs and NK cells, and this component of IL-12R plays a crucial role for IL-12-mediated activation of these cell types (19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30). Recently, murine splenic DCs exhibited transcriptional expression of IL-12Rß1 and IL-12Rß2 (37, 38), and IL-12 induced activation of these cells (37, 38, 39, 40). However, much is unknown about expression of IL-12Rß1 and IL-12Rß2 and the effect of IL-12 on human monocyte-derived DCs.
Flow cytometric analysis shows that IL-12Rß1 was expressed on
monocyte-derived DCs as well as TCs and Con A blasts, and the
expression level of IL-12Rß1 on monocyte-derived DCs was higher than
those of TCs and Con A blasts (Fig. 1
A). In contrast, little or no
expression of IL-12Rß1 were observed in monocytes (Fig. 1A).
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B
shows that IL-12Rß1 transcript was expressed in monocytes and
monocyte-derived DCs as well as TCs and Con A blasts.
We further examined the transcriptional expression of IL-12Rß2 in
TCs, Con A blasts, monocytes, and monocyte-derived DCs (Fig. 1B). Semiquantitative RT-PCR analysis revealed that the
transcript of IL-12Rß2 were expressed in TCs, Con A blasts, and DCs,
and the expression level of IL-12Rß2 in Con A blasts and DCs were
higher than that of TCs. We also observed little or no transcriptinal
expression of IL-12Rß2 in monocytes.
IL-12 induces production of various cytokine in DCs
To address the effect of IL-12 on the capacity of DCs to produce
cytokines, monocyte-derived DCs were unstimulated or stimulated with
IL-12 for 48 h, and the transcriptional levels of inducible
cytokines were examined by semiquantitative RT-PCR (Fig. 2). Stimulation of Con A blasts with
IL-12 caused the production of GM-CSF, IL-1ß, IL-6, TNF-,
IL-12p40, and IL-12p35 at transcriptional levels. In contrast,
treatment of monocyte-derived DCs with IL-12 resulted in the production
of GM-CSF, IL-1ß, IL-6, TNF-, and IL-12p40. We also observed that
the production level of IL-1ß in IL-12-stimulated monocyte-derived
DCs was higher than that of IL-12-stimulated Con A blasts, whereas the
stimulation of monocyte-derived DCs with IL-12 induced lower production
of IL-12p35 than that of IL-12-stimulated Con A blasts.
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production (39, 40). Therefore, we
examined the effect of IL-12 on the production of IFN- in
monocyte-derived DCs. The ligation by IL-12 induced the production of
the transcript of IFN- (Fig. 2) and its product (Table I) in these cells, and this productions
were comparable to those of IL-12-stimulated Con A blasts.
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The engagement of IL-12Rß1 by IL-12 increases the tyrosine
phosphorylation of targeted intracellular proteins in various cell
types, and these intracellular events appear to be crucial for their
functional activation (32, 33, 34, 35, 36). However, much less is
known about intracellular signaling events associated with protein
tyrosine phosphorylation cascades that are responsible for
IL-12-mediated activation of DCs. To address direct involvement of the
PTK-dependent pathway in the responsiveness for IL-12-mediated
functional activation of Con A blasts and monocyte-derived DCs, the
cells were unstimulated or stimulated with IL-12 (1–100 ng/ml), and
Western blots of whole-cell lysate stained with anti-pTyr mAb were
performed (Fig. 3A). Under
starved conditions, several intracellular proteins were weakly tyrosine
phosphorylated in unstimulated monocyte-derived DCs, while elevated
tyrosine phosphorylation appeared in various intracellular proteins in
these cells stimulated with IL-12 in a dose-dependent manner. We also
observed that IL-12 induced tyrosine phosphorylation events in Con A
blasts, and the pattern of these events were distinct from those of
monocyte-derived DCs.
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in response to IL-12. Therefore, we examined the role
of IL-12Rß1 in IL-12-induced tyrosine phosphorylation events in
monocyte-derived DCs. As shown in Fig. 3B,
anti-IL-12Rß1 mAb, but not cont. IgG, suppressed IL-12-induced
tyrosine phosphorylation of targeted intracellular proteins in
monocyte-derived DCs. We also observed that IL-12-induced tyrosine
phosphorylation of multiple proteins were suppressed by a mAb to
IL-12Rß1 in Con A blasts. These results indicate that IL-12-induced
tyrosine phosphorylation events are mainly mediated by IL-12Rß1 in
monocyte-derived DCs and Con A blasts.
We (30) have previously reported that the ligation by
IL-12 induced phosphorylation of tyrosine residues in the cytoplasmic
domain of IL-12Rß1 and its association with various
tyrosine-phosphorylated proteins in Con A blasts and
IL-12Rß1-transfected cells. To examine the tyrosine phosphorylation
level of IL-12Rß1 and its association with tyrosine-phosphorylated
proteins, the total cell lysates from monocyte-derived DCs and Con A
blasts unstimulated or stimulated with IL-12, were immunoprecipitated
with a mAb to IL-12Rß1, and were then subjected to Western blotting
with anti-pTyr mAb (Fig. 4,
A and B). We observed induction of tyrosine
phosphorylation of IL-12Rß1 in monocyte-derived DCs and Con A blasts
following stimulation with IL-12. The position of IL-12Rß1 (
100
kDa) were determined by Western blotting with a mAb to IL-12Rß1.
Furthermore, we found significant increase in the level of
tyrosine phosphorylation of various proteins associated with IL-12Rß1
from IL-12-stimulated Con A blasts and monocyte-derived DCs, while
these tyrosine-phosphorylated IL-12Rß1-associated proteins were
different between Con A blasts and monocyte-derived DCs. These results
indicate that IL-12 induces tyrosine phosphorylation of IL-12Rß1 as
well as recruitment of tyrosine-phosphorylated proteins to IL-12Rß1
in Con A blasts and monocyte-derived DCs.
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Previous studies have shown that the engagement by IL-12 is
associated with phosphorylation of two member of the Jak family (Jak2
and Tyk2) and Stat family (Stat3 and Stat4) (32, 33, 34, 35, 36). To
address whether these proteins were directly associated with
tyrosine-phosphorylated IL-12Rß1, the total cell lysates were
immunoprecipitated with IL-12Rß1 and were subjected to Western
blotting with respective Abs (Fig. 4, C–F). We found
Jak2/Tyk2 and Stat3/Stat4 proteins in the immunoprecipitates with
IL-12Rß1 in monocyte-derived DCs and Con A blasts following
stimulation with IL-12, whereas little or no detection of these
proteins in the immunoprecipitates with IL-12Rß1 in unstimulated
these cell types. These results indicate that ligation by IL-12 induced
association of Jak2/Tyk2 and Stat3/Stat4 with tyrosine-phosphorylated
IL-12Rß1.
IL-12 induces activation of Jak2/Tyk2 and Stat3/Stat4 in DCs
The ligation by IL-12 initiates tyrosine phosphorylation of Jak2
and Tyk2, which leads to their enzymatical activation in TCs and NK
cells (32, 33, 34, 35, 36). To clarify the potential involvement of
Jak2 and Tyk2 in IL-12-mediated activation of monocyte-derived DCs, the
cells were unstimulated or stimulated with IL-12, and the level of
their phosphorylation was assessed by immunoblotting with
anti-pTyr mAb (Fig. 5,
A–D). Stimulation of monocyte-derived DCs increased
tyrosine phosphorylation of Jak2 and Tyk2 compared with those of
unstimulated cells, and similar results were observed in Con A blasts.
In contrast, the total amounts of these Jaks were unchanged following
stimulation in these cell types (Fig. 5, A–D).
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, A–D). Indeed, the engagement by
IL-12 triggered tyrosine phosphorylation of Stat3 and Stat4 in
monocyte-derived DCs as well as Con A blasts, while the total amounts
of these Stat proteins were equivalent in unstimulated or
IL-12-stimulated cell types. These results indicate that IL-12 induces
Jak-dependent signaling events in monocyte-derived DCs and Con A
blasts. IL-12 induces activation of p38mapk in DCs
It has been shown that a cascade involving
p38mapk is required for IFN- production of
certain cell types (40). To test the potential involvement
of p38mapk in the IL-12-induced activation of
human DCs, monocyte-derived DCs and Con A blasts were unstimulated or
stimulated with IL-12, and the levels of tyrosine phosphorylation and
kinase activity of p38mapk were examined (Fig. 6). Stimulation IL-12 with
monocyte-derived DCs as well as Con A blasts increased tyrosine
phosphorylation and kinase activity of p38mapk
compared with unstimulated cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Grohmann et al. (37, 38) have recently reported that the
transcripts of IL-12Rß1 and IL-12Rß2 were more highly expressed in
murine splenic CD8- DCs than its
CD8+ counterpart. We showed that the
transcripts of IL-12Rß1 and IL-12Rß1 and the product of IL-12Rß1
on the cell surface were constitutively expressed in monocyte-derived
CD4+DCs as well as Con A blasts (Fig. 1). In
contrast, monocytes expressed IL-12Rß1, but not IL-12Rß2, at the
transcriptional level, whereas cell-surface expression of IL-12Rß1
was not observed in this cell type (Fig. 1). We also failed to detect
IL-12Rß1 products in monocytes by Western blotting with its mAb (data
not shown). Although monocytes did not express IL-12Rß1 on their cell
surface, stimulation with GM-CSF plus IL-4 may regulate
posttranscriptional expression of IL-12Rß1 in the differentiation of
DCs from monocytes. Further study will test this possibility.
Previous studies have shown that DCs exhibited the capacity to produce
various sets of cytokines in response to several extracellular stimuli
(18, 37, 39, 40, 42, 43, 44, 45). We showed that stimulation of
monocyte-derived DCs as well as Con A blasts with IL-12 induced the
production of the GM-CSF, IL-1ß, IL-6, TNF-, and IFN- (Fig. 2
and Table I). These result suggest that IL-12-induced DC-derived
cytokine production may contribute to the activation of TCs as well as
NK cells, B cells, and monocytes/macrophages to regulate immune
responses.
Grohmann et al. (37) have shown that IL-12 induced
endogenous production of IL-12 in splenic DCs in the murine system. We
showed that exogenous stimulation of human monocyte-derived DCs with
IL-12 induce the endogenous production of the IL-12p40 transcript, but
not the IL-12p35 transcript, while Con A blasts produce both
transcripts following exogenous stimulation with IL-12 (Fig. 2).
D’Andrea et al. (20) have shown that activated PBMC
preferentially produce 10- to 50-fold excess of IL-12p40 as compared
with IL-12p35. Therefore, stimulation with exogenous IL-12 may be
insufficient to produce IL-12p35 in Con A blasts and DCs. In contrast,
Ling et al. (22) have reported that the IL-12p40 homodimer
competed with the IL-12p70 heterodimer for the binding with IL-12
binding sites and suppressed the IL-12-induced proliferation of PHA
blasts (22). Thus, these phenomena led us to hypothesize
that IL-12-induced autocrine production of the p40 homodimer regulate
IL-12-induced activation of DCs, which lead to the termination of TC
activation during Ag presentation.
Previous studies have reported that the engagement by IL-12 initiates
tyrosine phosphorylation of various intracellular proteins in Con A
blasts and NK cells, events involved in IL-12-mediated functional
activation of these cell types (19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30). We show that
stimulation of monocyte-derived DCs induce tyrosine phosphorylation
events, and the pattern of phosphorylated protein is distinct from
those of Con A blasts (Fig. 3A). Furthermore, a mAb to
IL-12Rß1 abrogated IL-12-induced tyrosine phosphorylation events
(Fig. 3B). Although IL-12Rß1-mediated signaling events may
be mediated through a protein tyrosine phosphorylation cascade in DCs
and Con A blasts, distinct PTKs may be activated following stimulation
of IL-12 in these cell types.
Accumulating results showed that the activation of the cascades of
Jak/Stat and p38mapk are essential for
IL-12-mediated activation of Con A blasts, NK cells, neutrophils, and
DCs (28, 29, 30, 31, 32, 33, 34, 35, 36, 40). We showed that the ligation of
IL-12Rß1 by IL-12 induced tyrosine phosphorylation of the
intracellular domain of IL-12Rß1 as well as its recruitment of
Jak2/Tyk2 and Sta3/Stat4 (Fig. 4). Furthermore, this stimulation caused
the activation of the cascades of Jak/Stat (Fig. 5) and
p38mapk (Fig. 6). These results suggest that
activation of the cascades of Jak/Stat and
p38mapk may be involved in IL-12-mediated
functional activation of monocyte-derived DCs.
In summary, our results suggest that the endogenous secretion of IL-12 in DCs may regulate themselves as well as other cell types via production of various cytokines to control the homeostasis of the immune responses. Recently, IL-12 gene-transduced DCs have been shown to be effective for the initiation and the persistence of tumor-specific immunity (46). Defining the precise mechanisms by which DCs act on the regulation of TC activation during Ag presentation may provide further insight into the role of these cells in immune-related diseases and facilitate the use of DCs in vaccinations for cancer treatment.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: DC(s), dendritic cell(s); cont. IgG, control IgG; ECL, enhanced chemiluminescence; Jak, Janus kinase; MAPK(s), mitogen-activated protein kinase(s); PTK, protein tyrosine kinase; pTyr, phosphotyrosine; TC(s), T cell(s); CD40L, CD40 ligand; PVDF, polyvinylidene difluoride.
Received for publication August 31, 1999. Accepted for publication April 21, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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cooperate in the generation of dendritic Langerhans cells. Nature 360:258.[Medline]
. J. Exp. Med. 179:1109.-induced activation and maturation of human peripheral blood monocyte-derived dendritic cells. J. Immunol. 162:3865.-production by suppressing natural killer cell stimulatory factor/IL-12 synthesis in accessory cells. J. Exp. Med. 178:1041. and interleukin-4 regulate T cell interleukin-12 responsiveness through the differential modulation of high-affinity interleukin-12 receptor expression. E. J. Immunol. 27:647.[Medline]
. J. Immunol. 157:126.[Abstract]
B and primes DC for IL-12 production. Immunity 9:315.[Medline]
- dendritic cells to enhance presentation of a tumor peptide in vivo. J. Immunol. 163:3100. production by CD8+ dendritic cells. J. Exp. Med. 189:1981. production by dendritic cells. J. Immunol. 164:64.. J. Immunol. 160:1139.This article has been cited by other articles:
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 P.-H. Chiang, L. Wang, C. A. Bonham, X. Liang, J. J. Fung, L. Lu, and S. Qian Mechanistic Insights into Impaired Dendritic Cell Function by Rapamycin: Inhibition of Jak2/Stat4 Signaling Pathway J. Immunol., February 1, 2004; 172(3): 1355 - 1363. [Abstract] [Full Text] [PDF]
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S.-J. Ha, D.-J. Kim, K.-H. Baek, Y.-D. Yun, and Y.-C. Sung IL-23 Induces Stronger Sustained CTL and Th1 Immune Responses Than IL-12 in Hepatitis C Virus Envelope Protein 2 DNA Immunization J. Immunol., January 1, 2004; 172(1): 525 - 531. [Abstract] [Full Text] [PDF]
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R. Gutzmer, K. Langer, S. Mommert, M. Wittmann, A. Kapp, and T. Werfel Human Dendritic Cells Express the IL-18R and Are Chemoattracted to IL-18 J. Immunol., December 15, 2003; 171(12): 6363 - 6371. [Abstract] [Full Text] [PDF]
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 K. G. Hogg, S. Kumkate, and A. P. Mountford IL-10 regulates early IL-12-mediated immune responses induced by the radiation-attenuated schistosome vaccine Int. Immunol., December 1, 2003; 15(12): 1451 - 1459. [Abstract] [Full Text] [PDF]
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 D. Durali, M.-G. de Goer de Herve, J. Giron-Michel, B. Azzarone, J.-F. Delfraissy, and Y. Taoufik In human B cells, IL-12 triggers a cascade of molecular events similar to Th1 commitment Blood, December 1, 2003; 102(12): 4084 - 4089. [Abstract] [Full Text] [PDF]
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 J. E. A. Portielje, C. H. J. Lamers, W. H. J. Kruit, A. Sparreboom, R. L. H. Bolhuis, G. Stoter, C. Huber, and J. W. Gratama Repeated Administrations of Interleukin (IL)-12 Are Associated with Persistently Elevated Plasma Levels of IL-10 and Declining IFN-{gamma}, Tumor Necrosis Factor-{alpha}, IL-6, and IL-8 Responses Clin. Cancer Res., January 1, 2003; 9(1): 76 - 83. [Abstract] [Full Text] [PDF]
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M. L. Belladonna, J.-C. Renauld, R. Bianchi, C. Vacca, F. Fallarino, C. Orabona, M. C. Fioretti, U. Grohmann, and P. Puccetti IL-23 and IL-12 Have Overlapping, but Distinct, Effects on Murine Dendritic Cells J. Immunol., June 1, 2002; 168(11): 5448 - 5454. [Abstract] [Full Text] [PDF]
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C. Parham, M. Chirica, J. Timans, E. Vaisberg, M. Travis, J. Cheung, S. Pflanz, R. Zhang, K. P. Singh, F. Vega, et al. A Receptor for the Heterodimeric Cytokine IL-23 Is Composed of IL-12R{beta}1 and a Novel Cytokine Receptor Subunit, IL-23R J. Immunol., June 1, 2002; 168(11): 5699 - 5708. [Abstract] [Full Text] [PDF]
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T. K. Varma, T. E. Toliver-Kinsky, C. Y. Lin, A. P. Koutrouvelis, J. E. Nichols, and E. R. Sherwood Cellular Mechanisms That Cause Suppressed Gamma Interferon Secretion in Endotoxin-Tolerant Mice Infect. Immun., September 1, 2001; 69(9): 5249 - 5263. [Abstract] [Full Text] [PDF]
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A. K. Wesa and A. Galy IL-1{beta} induces dendritic cells to produce IL-12 Int. Immunol., August 1, 2001; 13(8): 1053 - 1061. [Abstract] [Full Text] [PDF]
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M. P. Rudolf, S. C. Fausch, D. M. Da Silva, and W. M. Kast Human Dendritic Cells Are Activated by Chimeric Human Papillomavirus Type-16 Virus-Like Particles and Induce Epitope-Specific Human T Cell Responses In Vitro J. Immunol., May 15, 2001; 166(10): 5917 - 5924. [Abstract] [Full Text] [PDF]
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T. Fukao, D. M. Frucht, G. Yap, M. Gadina, J. J. O'Shea, and S. Koyasu Inducible Expression of Stat4 in Dendritic Cells and Macrophages and Its Critical Role in Innate and Adaptive Immune Responses J. Immunol., April 1, 2001; 166(7): 4446 - 4455. [Abstract] [Full Text] [PDF]
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H. Schindler, M. B. Lutz, M. Rollinghoff, and C. Bogdan The Production of IFN-{{gamma}} by IL-12/IL-18-Activated Macrophages Requires STAT4 Signaling and Is Inhibited by IL-4 J. Immunol., March 1, 2001; 166(5): 3075 - 3082. [Abstract] [Full Text] [PDF]
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