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B1
Center for Immunology and Cancer Research, University of Queensland, Princess Alexandra Hospital, Brisbane, Queensland, Australia
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B, and particularly RelB, is an essential control
pathway for myeloid DC differentiation. Furthermore, RelB regulates B
cell Ag-presenting function. We hypothesized that CD40 ligand (CD40L)
and TNF-
, which differ in their capacity to condition DC, would also
differ in their capacity to activate NF-B. DC differentiated for 2
days from monocytes in the presence of GM-CSF and IL-4 were used as a
model, as NF-B activity was constitutively low. The capacity of DC
to activate T cells following CD40L treatment was enhanced compared
with TNF- treatment, and this was NF-B dependent. Whereas
RelB/p50 translocation induced by TNF- was attenuated after 6
h, RelB/p50 nuclear translocation induced by CD40L was sustained for at
least 24 h. The mechanism of this difference related to enhanced
degradation of IB following CD40L stimulation. However, NF-B
activation induced by TNF- could be sustained by blocking autocrine
IL-10. These data indicate that NF-B activation is essential for T
cell activation by DC, and that this function is enhanced if DC NF-B
activation is prolonged. Because IL-10 moderates DC NF-B activation
by TNF-, sustained NF-B activation can be achieved by blocking
IL-10 in the presence of stimuli that induce
TNF-. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Because the presentation of Ag
by immature DC may lead to tolerance, an understanding of the key
elements controlling the DC maturation program is critical for
designing therapies that effectively induce tumor-specific CTL and T
cell help despite the potential for attenuation of DC maturation in
environments such as tumors. Previously it was demonstrated that
ligation of CD40 expressed by DC conditioned the DC for effective APC
function, particularly CTL induction (6, 7, 8). Indeed,
activation with anti-CD40 Ab could substitute for T cell help when
inducing CTL in vivo. While the full range of signals that can
condition DC in this way is not known, influenza viral particles could
also condition DC for CTL induction (7). In keeping with
the importance of CD40 ligation in the induction of CTL effectors,
tumor Ag-pulsed bone marrow-derived DC treated ex vivo with
soluble CD40L led to enhanced tumor protection compared with DC
pretreated without soluble CD40L or treated with LPS
(9).
The NF-B family has emerged as a key transducer of inflammatory
signals to the DC maturation program. The NF-B complex comprises
homodimers and heterodimers of the structurally related proteins p50,
p52, RelA (p65), c-Rel, and RelB. NF-B proteins are present in an
inactive form in the cytosol, bound to inhibitor proteins called IB.
Signaling through NF-B-inducing kinase and other kinases induces
phosphorylation of IB and nuclear translocation of NF-B
(10, 11). RelB is required for DC differentiation, as
RelB-/- mice have normal numbers of peripheral
immature DC such as Langerhans cells but lack mature myeloid DC
(12, 13, 14). RelB, p50, and c-Rel are the major NF-B
transcription factor subunits induced during human monocyte-derived DC
(MDDC) and murine Langerhans cell maturation (15, 16). We have shown that RelB/p50 nuclear translocation
correlates with efficient APC function and directly enhances APC
function of B cells through regulation of CD40 and MHC expression
(17, 18).
Because NF-B, and particularly RelB, is a critical control pathway
for myeloid DC differentiation, we hypothesized that proinflammatory
signals that differ in their capacity to condition DC for CTL induction
would also differ in their capacity to activate NF-B. DC
differentiated for 2 days from monocytes in the presence of GM-CSF and
IL-4 (MDDC) were used as a model in these studies because their
constitutive activation of NF-B is low, and because MDDC are used in
many current and published trials of tumor immunotherapy using DC. The
data indicate that CD40L signals DC through potent and sustained
NF-B stimulation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Monocytes were derived from PBMC depleted of NK, B, and T cells
with anti-CD56, anti-CD16, anti-CD19, and anti-CD3, as
well as goat anti-mouse Ig-conjugated magnetic beads (Miltenyi
Biotec, Auburn, CA) as previously described (19). MDDC
were cultured for 2 days in medium supplemented with 800 U/ml GM-CSF
and 400 U/ml recombinant human IL-4 (both from Schering-Plough, Sydney,
Australia). To induce maturation, MDDC were incubated with 100 U/ml
TNF- (Life Technologies, Gaithersburg, MD), 500 ng/ml CD40L
(Immunex, Seattle, WA), 1 µg/ml LPS (Sigma-Aldrich, St. Louis, MO),
or 10 ng/ml IL-12 (R&D Systems, Minneapolis, MN). In some experiments
MDDC were incubated for 30 min with BAY 11-7082 (BioMol, Plymouth
Meeting, PA) and then TNF- was added for an additional 24 h.
MDDC were then washed three times before use. Neutralizing Abs
anti-IL-10 (BD PharMingen, San Diego, CA) and anti-IL-12 (BD
PharMingen) were used at 10 µg/ml. Control Abs were rat Ig and mouse
Ig, respectively.
Flow cytometry
MDDC were stained with mAb to HLA-DR-FITC (DK22; DAKO, Carpinteria, CA), CD83 (Ancell, Bayport, MN), or CD86 (Ancell). DC labeled with unconjugated Abs against CD83 and CD86 were subsequently incubated with biotinylated rabbit anti-mouse Ig (DAKO) and then with streptavidin-FITC (DAKO). Cells were analyzed using a Coulter Epics Elite flow cytometer (Coulter Electronics, Hialeah, FL).
Cytokine ELISA
Levels of IL-10 and IL-12 p70 in supernatants from MDDC
stimulated with TNF-, CD40L, or LPS were determined using OptEIA
kits (BD PharMingen) as per the manufacturer’s instructions.
Assays of APC function
MDDC were treated with mitomycin C (Sigma-Aldrich) as previously described (19). For the MLR, allogeneic T cells were purified from nylon wool nonadherent PBMC derived from healthy donors by negative selection with anti-CD19, anti-CD16, anti-CD56, anti-HLA-DR, and goat anti-mouse Ig-conjugated magnetic beads (Miltenyi Biotec) as previously described (17). Varying numbers of APC were incubated with 105 allogeneic T cells for 5 days. [3H]Thymidine (1 µCi/well; ICN Pharmaceuticals, Costa Mesa, CA) was added for the last 18 h. Plates were harvested using an automated harvester and counted using a Packard TopCount NXT (Packard Instrument, Meriden, CT). Results are expressed as mean ± SEM of triplicate wells.
Protein extraction and immunoblotting
Nuclear and cytoplasmic extracts were prepared as previously
described (17), and protein estimations were conducted
using a Protein Assay kit (Bio-Rad, Hercules, CA). A total of 10 µg
of protein extract were separated by 8% SDS-PAGE. Following transfer
to nitrocellulose (Amersham Biosciences, Sunnyvale, CA), membranes were
immunoblotted with anti-RelB (sc-226), anti-p50 (sc-7178),
anti-IB (sc-371) (all from Santa Cruz Biotechnology, Santa
Cruz, CA), or anti-phospho-IB (Ser32;
Cell Signaling Technology, Beverly, MA) Abs followed by sheep
anti-rabbit HRP-conjugated Ig (SILENUS Labs, Boronia, Australia)
and then detected by ECL according to the manufacturer’s instructions
(Amersham Biosciences). For protein quantitation, blots were scanned
using a Storm 860 (Amersham Biosciences) and analyzed by densitometry
using ImageQuant 5.1 software (Amersham Biosciences).
NF-B-binding ELISA
p50, RelB, and RelA DNA binding was detected by ELISA using a
Mercury Transfactor p50 kit (Clontech Laboratories, Palo Alto, CA). A
total of 10 µg of nuclear extract were bound to wells coated with
NF-B consensus oligonucleotide, then incubated with anti-RelB,
anti-p50, or anti-RelA, followed by anti-rabbit
HRP-conjugated Ig, and then detected by measuring color development of
tetramethylbenzidine at 650 nm using a Multiskan plate reader
(Labsystems, Chicago, IL).
RNA isolation and real-time quantitative PCR
RNA was extracted from 1 x 106 MDDC
using TRIzol (Life Technologies) according to the manufacturer’s
instructions. cDNA was synthesized using
oligo(dT)20 (Promega, Madison, WI) as a primer
and Expand Reverse Transcriptase (Boehringer Mannheim, Ridgefield, CT).
Primers were designed using Primer Express Software (PE Applied
Biosytems, Foster City, CA) and were as follows: GAPDH,
5'-GAAGGTGAAGGTCGGAGTC-3' and 5'-GAAGATGGTGATGGGATTTC-3'; RelB,
5'-GCCATTGCCTTTCACGTACCT-3' and 5'-CCCGTTTCGCCTTCTTGTC-3'; p50,
5'-GGCTACACCGAAGCAATTGAAG-3' and 5'-CAGCGAGTGGGCCTGAGA-3'; p52,
5'-CCGATTTCGATATGGCTGTGA-3' and 5'-GGTCTTTCGGCCCTTCTCA-3'; c-Rel,
5'-CCCACGCTCAGGCAATACA-3' and 5'-GGTGGGATACCTTGCGAATTAG-3'; RelA,
5'-CTGCCGGGATGGCTTCTAT-3' and 5'-CCAGGTTCTGGAAACTGTGGAT-3'. PCR
contained 100 pmol of each primer and 1x SYBR Green Master Mix (PE
Applied Biosystems) in a 25-µl volume. PCR cycling conditions were
95°C for 15 s and 60°C for 1 min for 40 cycles and conducted
on an ABI PRISM 7700 thermal cycler (PE Applied Biosystems). Relative
standard curves were generated for GAPDH, RelB, p50, p52, c-Rel, and
RelA by plotting dilutions of B cell lymphoblastoid cell line
cDNA of known RNA concentration against cycle threshold values. Using
cycle threshold values obtained from MDDC cDNA and standard curves
generated above, the amount of input RNA for GAPDH, RelB, p50, p52,
c-Rel, and RelA was determined. By normalizing for different GAPDH
input RNA levels, the relative amounts of NF-B mRNA were determined
and expressed as fold increase. Data are expressed as the mean ±
SEM of two independent experiments using different donor MDDC.
Statistical analysis
Differences were analyzed using paired t tests.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and CD40L differentially signal NF-B-dependent APC
function
CD40L and TNF- induce DC maturation but induce different
functional outcomes. Whereas TNF- induces reversible maturation of
MDDC, low levels of IL-12 secretion, and weak CTL induction, CD40L is a
potent signal for IL-12 production and CTL activation. We hypothesized
that CD40L and TNF- would also differ in their capacity to activate
DC NF-B. DC differentiated for 2 days from monocytes in the presence
of GM-CSF and IL-4 (MDDC) were used as a model. Two-day MDDC expressed
low levels of CD14, CD83, and CD86, moderate CD40, and high levels of
CD11c and HLA-DR (Fig. 1
A).
Constitutive RelB DNA binding activity was low (Fig. 1B),
and their capacity to stimulate allogeneic T cell proliferation was
modest (Fig. 1C). MDDC were stimulated for 24 h with
increasing doses of TNF- or CD40L and compared for nuclear RelB DNA
binding activity by ELISA. Although both CD40L and TNF- up-regulated
DNA binding of RelB, CD40L induced higher levels of RelB DNA binding
within the nucleus (Fig. 1B). Maximal RelB DNA binding
activity was induced with 100 U/ml TNF- and 500 ng/ml CD40L.
Increased levels of NF-B in CD40L-stimulated MDDC correlated with an
enhanced ability of these cells to stimulate allogeneic T cells
compared with DC treated with TNF- (Fig. 1C) and higher
levels of CD83, CD86, and HLA-DR surface expression (Fig. 2B). In summary, these data
show a close correlation between RelB DNA binding activity and MDDC APC
function.
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B signaling pathway to TNF--
and CD40L-induced MDDC maturation, the NF-B pathway was inhibited at
the time of maturation induction. The compound BAY 11-7082 has
previously been shown to block NF-B induction in endothelial cells
through the inhibition of IB phosphorylation (20).
MDDC were treated for 24 h with 100 U/ml TNF- or 500 ng/ml
CD40L, as these doses had been shown in the previous experiments to
maximally induce RelB DNA binding and APC function. Treatment in the
presence of BAY 11-7082 blocked DNA binding of RelB, p50, and RelA in a
dose-dependent manner, with 10 µM effectively inhibiting NF-B DNA
binding (Fig. 2
A). Consistent with the previously reported
inhibitory effects of BAY 11-7082 on IB processing, BAY 11-7082
inhibited TNF--induced nuclear translocation but not cytoplasmic
expression of RelB and p50 (data not shown). BAY 11-7082 blocked CD40L
and TNF--induced up-regulation of CD86, HLA-DR, and CD83 in a
dose-dependent manner (Fig. 2B), although TNF- induced
lower levels of CD83 and HLA-DR than did CD40L. In addition,
10 µM BAY 11-7082 inhibited the up-regulation of DC APC
function induced by CD40L or TNF- (Fig. 2C). The data
indicate that CD40L stimulation of MDDC induces higher levels of
NF-B DNA binding compared with TNF-, and this correlates with
enhanced APC function. Moreover, blockade of the NF-B pathway is
sufficient to completely prevent TNF-- or CD40L-induced MDDC
maturation associated with enhanced capacity for T cell activation.
CD40L signals sustained activation of NF-B in MDDC
Because NF-B activation is required for functional maturation
of MDDC, signals that enhance the APC function of DC most effectively
would be expected to have potent effects on NF-B. To compare
activation by CD40L and TNF- in more detail, the kinetics of NF-B
induction were examined. Nuclear p50 and RelB were assessed by
immunoblotting. Incubation of MDDC with 100 U/ml TNF- or 500 ng/ml
CD40L induced nuclear translocation of RelB and p50 in MDDC within 30
min (Fig. 3). Whereas TNF- induced
maximal nuclear translocation of RelB and p50 at 4 h, nuclear
translocation continued for at least 24 h after stimulation with
CD40L. In keeping with the dose-response curves shown in Fig. 1, neither 500 nor 1000 U/ml TNF- increased the nuclear translocation
of RelB after 4 h (data not shown).
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B induction by CD40L
Sustained transcriptional activation of RelB, p50, and c-Rel.
The next series of experiments explored the mechanism behind the
sustained induction of NF-B by CD40L as opposed to TNF-. We
demonstrated previously that RelB activity in DC was regulated both
transcriptionally and through nuclear translocation upon DC activation
(17). To test whether TNF- or CD40L also induced
NF-B mRNA in MDDC, levels of NF-B mRNA were quantitatively
analyzed by real-time PCR following activation. TNF- signaled a
2-fold increase in RelB, c-Rel, and p50 mRNA within 30 min, increasing
4- to 5-fold by 2 h (Fig. 4).
Whereas RelB mRNA levels remained elevated for 24 h, c-Rel and p50
mRNA levels returned almost to baseline levels by 24 h. The
induction of RelB, c-Rel, and p50 mRNA by CD40L was slower than that
induced by TNF-, in that a 2-fold increase occurred within 1–2 h,
and mRNA levels continued to increase for 6–24 h (Fig. 4). Neither
RelA nor p52 mRNA was induced by either stimulus (Fig. 4 and data not
shown). The data indicate that signaling by either TNF- or CD40L
induces rapid nuclear translocation of RelB and p50 in 48-h MDDC. This
translocation is accompanied by increased RelB, p50, and c-Rel mRNA.
Sustained RelB/p50 translocation following CD40L treatment of DC is
associated with a sustained increase in RelB, p50, and c-Rel
mRNA.
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B.
Because retention of NF-B in the cytoplasm by inhibitory proteins
(IB) is essential to NF-B regulation, the attenuated nuclear
translocation of NF-B induced by TNF- may be a consequence of
increasing cytoplasmic retention by IB. To test this, protein levels
of the predominant IB, which has been shown to inhibit all
NF-B subunits, were examined in cytoplasmic extracts of MDDC
stimulated with TNF- or CD40L. Whereas TNF- induced increasing
cytoplasmic IB from 1 h onwards, CD40L induced maximal
IB at 2 h that declined up until 24 h (Fig. 5). The decline in IB following
CD40L stimulation coincided with the appearance of phosphorylated
IB (Fig. 5A) and strong nuclear RelB/p50 location
(Fig. 3). Because phosphorylated IB is actively degraded, the
data indicate that, although IB is induced by CD40L treatment,
active proteolysis prevents IB accumulation.
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is inhibitory for NF-B.
TNF- stimulation of monocytes induces IL-10, and LPS and other
signals stimulate autocrine IL-10 production by immature MDDC that can
inhibit maturation, IL-10, and IB (21, 22). Using the
previous model, the next experiments tested whether IL-10 regulated
nuclear RelB translocation by TNF-. Initially, levels of IL-10 in
supernatants of MDDC stimulated for 24 h in the presence of
TNF-, LPS, or CD40L were determined. Whereas stimulation of MDDC
with CD40L failed to induce IL-10, both TNF- and LPS stimulation
induced IL-10 by 
5- and 10-fold, respectively (Table I). Inhibition of IL-10 by addition of
anti-IL-10 neutralizing Ab enhanced TNF--induced RelB
translocation at 24 h (Fig. 6, A and B). In addition, anti-IL-10 increased
RelB DNA binding activity and APC function of TNF--stimulated MDDC
to levels similar to those observed for CD40L-stimulated MDDC (Fig. 6, A and B). These effects were mediated via
NF-B, as addition of 10 µM Bay 11-7082 reversed the effects of
IL-10 neutralization on RelB DNA binding and APC function (Figs. 6, C and D). These data indicate that autocrine
IL-10 is sufficient to attenuate NF-B activation of MDDC in response
to TNF-.
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B by CD40L rather than downstream
IL-12 production.
Twenty-four-hour stimulation of MDDC with TNF-, CD40L, or LPS
induced 3-, 9-, and 40-fold higher levels of IL-12 p70 produced in
supernatant compared with unstimulated MDDC (Table I). To determine
whether IL-12 produced in response to CD40L might influence RelB
nuclear translocation, anti-IL-12 neutralizing Ab was added to MDDC
in the presence of TNF- or CD40L. Inhibition of IL-12 had no effect
on TNF--induced RelB translocation (Fig. 6, A and
B), modestly reduced CD40L-induced RelB translocation at
24 h (Fig. 6, A and B), and had no effect on
the APC function of DC in response to CD40L (Fig. 7A). While addition of
exogenous IL-12 to MDDC strongly induced RelB, p50, and c-Rel mRNA
after 24 h, RelB, p50, and RelA nuclear DNA binding was only
weakly induced (Figs. 7, B and C).
Therefore, although exogenous IL-12 can induce NF-B activation, this
induction is weak, and IL-12 produced in response to CD40L contributes
to MDDC NF-B translocation only minimally, at least at 24
h.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B pathway is
sufficient to prevent MDDC maturation, induced by maximal doses of
TNF- or CD40L, and associated enhanced capacity for T cell
activation. Collectively, these data indicate that NF-B activation
is required for functional maturation of MDDC and strongly implicate
NF-B in the coordinate up-regulation of cell surface molecules
associated with enhanced APC function. These data reinforce previous
observations that the NF-B pathway is activated during DC maturation
by diverse signals such as microbial products, inflammatory cytokines,
and CD40L (15, 16, 27, 28, 29, 30, 31, 32), and that maturation of MDDC
can be blocked by overexpression of IB or by use of a
cell-permeable peptide to prevent NF-B nuclear localization
(27, 33). However, not all signals induce equivalent DC
activation, and as a result different proinflammatory signals translate
to different T cell outcomes. For example, TNF- induces reversible
DC maturation, low levels of IL-12, and weak CTL, while CD40L
stimulates high levels of IL-12 and is a key conditioning stimulus for
CTL activation (6, 7, 8).
Given that TNF- and CD40L induce maturation of DC through NF-B
activation but lead to different functional outcomes, the current
studies examined whether these signals differ in their capacity to
activate NF-B. Indeed, CD40L induced sustained high levels of
RelB/p50 activation in MDDC. This occurred by several mechanisms. In
peripheral blood DC precursors, RelB activity is regulated both
at the mRNA level and through nuclear translocation (17).
Stimulation of MDDC by TNF- and CD40L revealed striking differences
in RelB, p50, and c-Rel mRNA levels. RelB, p50, and c-Rel mRNA was
rapidly induced and rapidly attenuated in response to TNF-. In
contrast, RelB, p50, and c-Rel mRNA was induced by CD40L more slowly,
but higher levels of RelB and c-Rel were induced, and RelB mRNA was
increasing even 24 h later. The selective induction of RelB, p50,
and c-Rel mRNA implicates these subunits in NF-B transcriptional
autoregulation. Of interest, despite differences in the intensity with
each signal, NF-B activation in D1 cells stimulated with either live
bacteria or LPS showed similar attenuation at 24 h as observed
here for TNF- stimulation (29).
TNF- and CD40L also differed in their capacity to degrade IB.
IB proteins regulate NF-B through cytoplasmic retention. Although
five mammalian IB proteins have been characterized, IB is
likely to play a major role in repression of NF-B in MDDC. First,
IB is expressed at high levels by MDDC (16)
and can inhibit transactivation of all known NF-B subunit
combinations (34). Second, ectopic expression of IB
in MDDC can inhibit NF-B nuclear translocation (33).
However, other proteins, including p105 and p100, may also be important
(35). In the current studies cytoplasmic IB levels
increased several hours after CD40L or TNF- stimulation of MDDC.
Moreover, in the presence of CD40L for 24 h, hyperphosphorylated
IB appeared, suggesting that IB is actively degraded.
Together with the mRNA data, this suggests a further mechanism for the
sustained nuclear induction of RelB/p50 after CD40 ligation, in that
newly synthesized p50 and RelB could associate and translocate to the
nucleus.
Consistent with the increasing cytoplasmic expression of IB and
with the ability of IL-10 to inhibit IKK activity and NF-B DNA
binding (36), the current studies also indicate that
autocrine IL-10, produced in response to TNF- but not CD40L,
moderates NF-B activity of MDDC. IL-10 has potent
anti-inflammatory effects ranging from induction of
anti-inflammatory soluble TNFR and IL-1R antagonist to
down-regulation of APC function (37, 38). Previously,
neutralization of IL-10 has been shown to enhance the capacity of 6-day
MDDC to stimulate T cell proliferation and IFN-
production following
LPS or CD40L signaling (22). In the current studies,
anti-IL-10 produced only modest enhancement of NF-B
translocation in response to CD40L and did not affect APC function.
These differences almost certainly relate to differences in IL-10
production by MDDC harvested at days 2 or 6. Moderation of DC APC
function by exogenous IL-10 in the presence of TNF- or CD40L has
been shown previously; however, due to reduced IL-10R expression upon
DC maturation, the effect of exogenous IL-10 varies depending on the
activation state of the DC at the time of IL-10 exposure (32, 39). In contrast to TNF-, CD40L provides a strong, sustained
NF-B activation signal to MDDC that is only somewhat dependent, at
24 h, on autocrine IL-12 signaling of NF-B. This is in keeping
with the relatively weak activation of NF-B by exogenous IL-12.
Although it is possible that IL-12 might have more prolonged effects on
DC differentiation than were examined in this work, it is more likely
that the major mechanisms for sustained NF-B translocation in
response to CD40L relate to positive transcriptional feedback and
enhanced IB phosphorylation. Taken together with functional
inhibition of MDDC by NF-B inhibitors, the data strongly support the
idea that NF-B plays a central role in transducing environmental
signals such as TNF-, LPS, IL-12, CD40L, and IL-10 for modulation of
DC function. Clearly, the data imply that the microenvironment of the
DC will have a major impact on its APC function.
Although both TNF- and CD40L activate NF-B through specific
receptors via common TNFR-associated factor and NF-B-inducing
kinases, the two stimuli differ in their ability to induce IL-10. The
mechanism behind selective IL-10 induction is unknown; however, it may
be through posttranscriptional regulation of IL-10, as transcription is
regulated by ubiquitous transcription factors Sp1 and Sp3 (40, 41). Irrespective of the mechanism of IL-10 induction, IL-10 has
the capacity to act in an autocrine manner to signal through the IL-10R
and to suppress nuclear activity of NF-B. Signaling via IL-10R
activates Janus kinase 1 and tyrosine kinase 2, as well as the
latent transcription factors STAT1 and STAT3, leading to inhibition of
proinflammatory cytokines, chemokines, and Ag-presenting function
(42). The likely mechanism of this inhibition is through
IL-10-induced transcription of suppressors of cytokine signaling-3
genes, which have proinflammatory attenuating functions
(43).
The current studies have important implications for design of tumor
immunotherapy. Conditioning DC with signals such as CD40 ligation or
viral infection is necessary for activation of CTL in lymph nodes
(7). The potent and sustained effect of CD40 ligation on
NF-B nuclear translocation shown in this work provides at least one
explanation for the exquisite sensitivity of CD40L for CTL activation
in draining lymph nodes, as NF-B, particularly RelB,
transcriptionally activates MHC class I and CD40 itself (18, 44). Similarly, DC treated with CD40L and pulsed with tumor Ag
enhance antitumor CTL and CD4+ T cell responses
(9). In contrast, immunosuppressive cytokines such as
IL-10, TGF-, and vascular endothelial growth factor (32, 45, 46) regulate DC maturation and function in tumor environments as
well as in normal skin, lung, or gut epithelium. These cytokines reduce
NF-B activity and contribute to the prevention of DC differentiation
in these environments. Therefore, NF-B is a central biochemical
pathway that regulates DC function in response to environmental
signals. This implies that signals transduced by NF-B in DC ex vivo
or in vivo will influence T cell responses in immunotherapy, and that
sustained activation of NF-B, particularly RelB, can be used as an
effective read-out of the functional status of a DC when designing
immunotherapeutic strategies.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Ranjeny Thomas, Center for Immunology and Cancer Research, University of Queensland, Princess Alexandra Hospital, Ipswich Road, Woolloongabba, Brisbane, Queensland 4102, Australia. E-mail address: rthomas{at}medicine.pa.uq.edu.au
3 Abbreviations used in this paper: DC, dendritic cell; CD40L, CD40 ligand; MDDC, monocyte-derived DC.
Received for publication December 26, 2001. Accepted for publication March 21, 2002.
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 C. Capini, M. Jaturanpinyo, H.-I Chang, S. Mutalik, A. McNally, S. Street, R. Steptoe, B. O'Sullivan, N. Davies, and R. Thomas Antigen-Specific Suppression of Inflammatory Arthritis Using Liposomes J. Immunol., March 15, 2009; 182(6): 3556 - 3565. [Abstract] [Full Text] [PDF]
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 C. J. Baglole, S. B. Maggirwar, T. A. Gasiewicz, T. H. Thatcher, R. P. Phipps, and P. J. Sime The Aryl Hydrocarbon Receptor Attenuates Tobacco Smoke-induced Cyclooxygenase-2 and Prostaglandin Production in Lung Fibroblasts through Regulation of the NF-{kappa}B Family Member RelB J. Biol. Chem., October 24, 2008; 283(43): 28944 - 28957. [Abstract] [Full Text] [PDF]
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Z. U. A. Mollah, S. Pai, C. Moore, B. J. O'Sullivan, M. J. Harrison, J. Peng, K. Phillips, J. B. Prins, J. Cardinal, and R. Thomas Abnormal NF-{kappa}B Function Characterizes Human Type 1 Diabetes Dendritic Cells and Monocytes J. Immunol., March 1, 2008; 180(5): 3166 - 3175. [Abstract] [Full Text] [PDF]
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 N. Ade, D. Antonios, S. Kerdine-Romer, F. Boisleve, F. Rousset, and M. Pallardy NF-{kappa}B Plays a Major Role in the Maturation of Human Dendritic Cells Induced by NiSO4 but not by DNCB Toxicol. Sci., October 1, 2007; 99(2): 488 - 501. [Abstract] [Full Text] [PDF]
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 K. P. A. MacDonald, R. D. Kuns, V. Rowe, E. S. Morris, T. Banovic, H. Bofinger, B. O'Sullivan, K. A. Markey, A. L. Don, R. Thomas, et al. Effector and regulatory T-cell function is differentially regulated by RelB within antigen-presenting cells during GVHD Blood, June 1, 2007; 109(11): 5049 - 5057. [Abstract] [Full Text] [PDF]
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M. Li, X. Zhang, X. Zheng, D. Lian, Z.-X. Zhang, W. Ge, J. Yang, C. Vladau, M. Suzuki, D. Chen, et al. Immune Modulation and Tolerance Induction by RelB-Silenced Dendritic Cells through RNA Interference J. Immunol., May 1, 2007; 178(9): 5480 - 5487. [Abstract] [Full Text] [PDF]
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L. Klotz, I. Dani, F. Edenhofer, L. Nolden, B. Evert, B. Paul, W. Kolanus, T. Klockgether, P. Knolle, and L. Diehl Peroxisome Proliferator-Activated Receptor {gamma} Control of Dendritic Cell Function Contributes to Development of CD4+ T Cell Anergy J. Immunol., February 15, 2007; 178(4): 2122 - 2131. [Abstract] [Full Text] [PDF]
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F. Moore, S. Buonocore, E. Aksoy, N. Ouled-Haddou, S. Goriely, E. Lazarova, F. Paulart, C. Heirman, E. Vaeremans, K. Thielemans, et al. An Alternative Pathway of NF-{kappa}B Activation Results in Maturation and T Cell Priming Activity of Dendritic Cells Overexpressing a Mutated I{kappa}B{alpha} J. Immunol., February 1, 2007; 178(3): 1301 - 1311. [Abstract] [Full Text] [PDF]
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E. Assier, V. Marin-Esteban, A. Haziot, E. Maggi, D. Charron, and N. Mooney TLR7/8 agonists impair monocyte-derived dendritic cell differentiation and maturation J. Leukoc. Biol., January 1, 2007; 81(1): 221 - 228. [Abstract] [Full Text] [PDF]
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P. Sun, C. M. Celluzzi, M. Marovich, H. Subramanian, M. Eller, S. Widjaja, D. Palmer, K. Porter, W. Sun, and T. Burgess CD40 Ligand Enhances Dengue Viral Infection of Dendritic Cells: A Possible Mechanism for T Cell-Mediated Immunopathology J. Immunol., November 1, 2006; 177(9): 6497 - 6503. [Abstract] [Full Text] [PDF]
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 Y.-L. Lin, S.-S. Lee, S.-M. Hou, and B.-L. Chiang Polysaccharide Purified from Ganoderma lucidum Induces Gene Expression Changes in Human Dendritic Cells and Promotes T Helper 1 Immune Response in BALB/c Mice Mol. Pharmacol., August 1, 2006; 70(2): 637 - 644. [Abstract] [Full Text] [PDF]
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 M. I. Iruretagoyena, S. E. Sepulveda, J. P. Lezana, M. Hermoso, M. Bronfman, M. A. Gutierrez, S. H. Jacobelli, and A. M. Kalergis Inhibition of Nuclear Factor-{kappa}B Enhances the Capacity of Immature Dendritic Cells to Induce Antigen-Specific Tolerance in Experimental Autoimmune Encephalomyelitis J. Pharmacol. Exp. Ther., July 1, 2006; 318(1): 59 - 67. [Abstract] [Full Text] [PDF]
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B. J. O'Sullivan, H. E. Thomas, S. Pai, P. Santamaria, Y. Iwakura, R. J. Steptoe, T. W. H. Kay, and R. Thomas IL-1beta Breaks Tolerance through Expansion of CD25+ Effector T Cells. J. Immunol., June 15, 2006; 176(12): 7278 - 7287. [Abstract] [Full Text] [PDF]
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B. Blanco, J. A. Perez-Simon, L. I. Sanchez-Abarca, X. Carvajal-Vergara, J. Mateos, B. Vidriales, N. Lopez-Holgado, P. Maiso, M. Alberca, E. Villaron, et al. Bortezomib induces selective depletion of alloreactive T lymphocytes and decreases the production of Th1 cytokines Blood, May 1, 2006; 107(9): 3575 - 3583. [Abstract] [Full Text] [PDF]
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A. D. Recklies, H. Ling, C. White, and S. M. Bernier Inflammatory Cytokines Induce Production of CHI3L1 by Articular Chondrocytes J. Biol. Chem., December 16, 2005; 280(50): 41213 - 41221. [Abstract] [Full Text] [PDF]
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M. O'Keeffe, R. J. Grumont, H. Hochrein, M. Fuchsberger, R. Gugasyan, D. Vremec, K. Shortman, and S. Gerondakis Distinct roles for the NF-{kappa}B1 and c-Rel transcription factors in the differentiation and survival of plasmacytoid and conventional dendritic cells activated by TLR-9 signals Blood, November 15, 2005; 106(10): 3457 - 3464. [Abstract] [Full Text] [PDF]
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 K. Vermaelen and R. Pauwels Pulmonary Dendritic Cells Am. J. Respir. Crit. Care Med., September 1, 2005; 172(5): 530 - 551. [Abstract] [Full Text] [PDF]
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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]
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 J. Bayry, S. Lacroix-Desmazes, V. Donkova-Petrini, C. Carbonneil, N. Misra, Y. Lepelletier, S. Delignat, S. Varambally, E. Oksenhendler, Y. Levy, et al. Natural antibodies sustain differentiation and maturation of human dendritic cells PNAS, September 28, 2004; 101(39): 14210 - 14215. [Abstract] [Full Text] [PDF]
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S. Bhattacharyya, P. Sen, M. Wallet, B. Long, A. S. Baldwin Jr, and R. Tisch Immunoregulation of dendritic cells by IL-10 is mediated through suppression of the PI3K/Akt pathway and of I{kappa}B kinase activity Blood, August 15, 2004; 104(4): 1100 - 1109. [Abstract] [Full Text] [PDF]
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A. G. Thompson, B. J. O'Sullivan, H. Beamish, and R. Thomas T Cells Signaled by NF-{kappa}B- Dendritic Cells Are Sensitized Not Anergic to Subsequent Activation J. Immunol., August 1, 2004; 173(3): 1671 - 1680. [Abstract] [Full Text] [PDF]
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 M. Movassagh, D. Laderach, and A. Galy Proteins of the Ikaros family control dendritic cell maturation required to induce optimal Th1 T cell differentiation Int. Immunol., June 1, 2004; 16(6): 867 - 875. [Abstract] [Full Text] [PDF]
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N. J. Mason, H.-C. Liou, and C. A. Hunter T Cell-Intrinsic Expression of c-Rel Regulates Th1 Cell Responses Essential for Resistance to Toxoplasma gondii J. Immunol., March 15, 2004; 172(6): 3704 - 3711. [Abstract] [Full Text] [PDF]
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J. G. Elliot, C. M. Jensen, S. Mutavdzic, J. P. Lamb, N. G. Carroll, and A. L. James Aggregations of Lymphoid Cells in the Airways of Nonsmokers, Smokers, and Subjects with Asthma Am. J. Respir. Crit. Care Med., March 15, 2004; 169(6): 712 - 718. [Abstract] [Full Text] [PDF]
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D. Frleta, R. J. Noelle, and W. F. Wade CD40-mediated up-regulation of Toll-like receptor 4-MD2 complex on the surface of murine dendritic cells J. Leukoc. Biol., December 1, 2003; 74(6): 1064 - 1073. [Abstract] [Full Text]
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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]
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D. Laderach, D. Compagno, O. Danos, W. Vainchenker, and A. Galy RNA Interference Shows Critical Requirement for NF-{kappa}B p50 in the Production of IL-12 by Human Dendritic Cells J. Immunol., August 15, 2003; 171(4): 1750 - 1757. [Abstract] [Full Text] [PDF]
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M. Hewison, L. Freeman, S. V. Hughes, K. N. Evans, R. Bland, A. G. Eliopoulos, M. D. Kilby, P. A. H. Moss, and R. Chakraverty Differential Regulation of Vitamin D Receptor and Its Ligand in Human Monocyte-Derived Dendritic Cells J. Immunol., June 1, 2003; 170(11): 5382 - 5390. [Abstract] [Full Text] [PDF]
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J. Xie, Y. Wang, M. E. Freeman III, B. Barlogie, and Q. Yi beta 2-Microglobulin as a negative regulator of the immune system: high concentrations of the protein inhibit in vitro generation of functional dendritic cells Blood, May 15, 2003; 101(10): 4005 - 4012. [Abstract] [Full Text] [PDF]
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K. Duperrier, A. Farre, J. Bienvenu, N. Bleyzac, J. Bernaud, L. Gebuhrer, D. Rigal, and A. Eljaafari Cyclosporin A inhibits dendritic cell maturation promoted by TNF-{alpha} or LPS but not by double-stranded RNA or CD40L J. Leukoc. Biol., November 1, 2002; 72(5): 953 - 961. [Abstract] [Full Text] [PDF]
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