CD40 Induces Interleukin-6 Gene Transcription in Dendritic
Cells
REGULATION BY TRAF2, AP-1, NF-
B, AND CBF1*
Jelena
Mann
§,
Fiona
Oakley,
Peter W. M.
Johnson§, and
Derek A.
Mann¶
From the Division of Infection, Inflammation and
Repair and § Division of Cancer Sciences, University of
Southampton, Southampton General Hospital,
Southampton SO16 6YD, United Kingdom
Received for publication, September 25, 2001, and in revised form, February 28, 2002
 |
ABSTRACT |
CD40-induced activation of cytokine gene
expression in dendritic cells (DC) is an important process in the
initiation of primary immune responses. We have determined the
intracellular signaling events that lead to CD40 ligation-induced
activation of interleukin-6 (IL-6) gene transcription in a murine DC
line, FSDC, that is phenotypically representative of bone
marrow-derived DC. IL-6 reverse transcriptase-PCR and promoter
assays established the responsiveness of FSDC to anti-CD40 ligation.
Further promoter assays showed that the transcription factors NF-B
and AP-1 are downstream transcriptional mediators of CD40-induced IL-6
gene expression. Anti-CD40 treatment of FSDC stimulated increased
expression of specific NF-B (p50:p65) and AP-1 (c-Jun, JunB, JunD,
and c-Fos) DNA-protein complexes. Overexpression of an IB-
super-repressor or a dominant negative JunD resulted in a strong
inhibition of CD40-inducible IL-6 promoter activity supporting a role
for both transcription factors. Upstream signal transduction events
were studied by transfection of wild type and mutant human CD40
expression constructs into FSDC followed by stimulation with an
anti-human CD40 antibody. These experiments revealed that anti-CD40
stimulation of NF-B and IL-6 gene transcription requires specific
amino acid residues in the cytoplasmic region of CD40 involved in the
recruitment of TRAF2. Induction of IL-6 mRNA by anti-CD40 treatment
was found to be a transient event (24 h) and was followed by a
diminution of IL-6 transcript to levels below those found in
unstimulated cells. This loss of IL-6 expression was associated with
reduced p50:p65 NF-B DNA binding and elevated binding of CBF1 to a
site overlapping the NF-B site. Overexpression of CBF1 resulted in a
profound inhibition of basal and anti-CD40-induced IL-6 promoter
activities indicating that prolonged induction of CBF1 may contribute
to the transient nature of the IL-6 response. The physiological
relevance of these molecular events to DC function is discussed.
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INTRODUCTION |
Dendritic cells (DCs)1
are antigen presenting cells that are specialized to have
the unique property of being able to prime naive T cells. The powerful
immunogenic properties of DCs have implicated these cells in the
abrogation of peripheral T cell tolerance against viral, microbial,
tumor, and transplant antigens (1). Following antigen capture the
immature DC undergoes phenotypic and functional changes that promote
its antigen presenting cells characteristics (1). Maturation events
involving migration to the lymph nodes, alteration of intracellular
major histocompatibility class II transport and surface expression, and
decreased capacity to take up and process antigen are required for the
DC to be able to activate T cells (1). Interaction of CD40 on DCs with
CD40 ligand (CD40L) on naive T cells is critical for DC maturation and
the generation of antigen-specific T cell responses (1, 2). CD40L
promotes DC survival, elevates expression of major histocompatibility
and co-stimulatory molecules, and induces the expression of a variety
of cytokines including tumor necrosis factor, IL-1, IL-6, IL-12, IL-15,
and IL-18, all of which are involved in T cell activation and
proliferation (1, 2).
IL-6 is a highly pleiotropic cytokine with properties that indicate it
is not only a stimulator of the activation, proliferation, and survival
of T cells (3-9), but is also able to modify DC function and survival
(1, 10, 11). A direct effect of IL-6 on the proliferation of T cells
has been documented by several groups (3-8), as has the ability of
IL-6 to prevent the death of naive T cells (9). Other studies have
described the ability of IL-6 to induce IFN
secretion in
differentiating T cells; indeed lack of this response in IL-6 gene
knockout mice renders animals unable to mount T cell responses against
Mycobacterium. tuberculosis and Toxoplasma gonddi
(12, 13). Production of IL-6 by CD40-stimulated DCs (1, 11) is
therefore likely to be important in promoting the generation and
survival of antigen-specific cytotoxic T cells. IL-6 can alter the
manner in which antigen is processed by DCs enabling the activation of
T cells against determinants that were previously cryptic (10). In
addition, Grohmann and colleagues (11) reported that autocrine IL-6
mediates most of the anti-tolerogenic effects of CD40 ligation on
CD8+ DC. Aberrant regulation of autocrine and paracrine
stimulation of DCs by IL-6 may therefore be important in the generation
of anti-self immunity and propagation of autoimmune disease.
The signal transduction events leading to the activation of cytokine
gene transcription by stimulation of CD40 have mainly been studied in B
cells. By contrast little is understood about these events in other
cell types, including DCs (2). Studies regarding the transcriptional
regulation of IL-6 in response to engagement of cell surface CD40 by
either CD40L or anti-CD40 have so far revealed a potential role for
NF-B and the requirement for amino acids 202 to 225 in the
cytoplasmic tail of CD40 (14). These amino acids in combination with
other motifs in the cytoplasmic tail of CD40 are responsible for the
recruitment of Janus kinase 3 (Jak3) and several members of the tumor
necrosis factor receptor-associated factor (TRAF) protein family (2,
14-21). These factors function to link CD40 to the signaling pathways
that activate NF-B, c-Jun NH2-terminal kinase (JNK), and
STAT3, which in turn stimulate transcription of various CD40-responsive
genes. Studies in a variety of cell types have shown that induction of
IL-6 gene transcription involves the coordinated regulation of factors
that associate with the evolutionary conserved AP-1, NF-B, and
NF-IL-6 sites in the mouse and human IL-6 gene promoters (22-27).
Similar detailed studies on the promoter and transcription factor
requirements for stimulation of IL-6 gene transcription following CD40
engagement are currently lacking. Since there is also a general
scarcity of information regarding the transcriptional regulation of DC function, we have therefore undertaken a detailed investigation into
events at both the IL-6 promoter and the cytoplasmic tail of CD40 that
mediate induction of IL-6 mRNA synthesis in response to CD40 engagement.
Using the murine DC line FSDC (28), we show that anti-CD40 treatment of
cells leads to a powerful, but transient induction of IL-6 mRNA
expression. We delineate the structural features of the IL-6 promoter
required for this response and in addition to demonstrating CD40
induction of AP-1 and NF-B DNA binding activities we also define a
role for specific members of these transcription factor families and
their inhibitors in the IL-6 response. Finally, co-transfection of the
IL-6 promoter with expression vectors for wild type or mutant human
CD40 proteins was employed to map amino acid residues in the
cytoplasmic tail of CD40 that are required for activation of NF-B
and induction of IL-6 mRNA synthesis.
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MATERIALS AND METHODS |
Cell Culture--
FSDC cell line (28), DC2.4 cell line (29), and
bone marrow-derived dendritic cells (BMDC) were cultured in RPMI 1640 medium, supplemented with 100 units/ml penicillin, 100 µg/ml
streptomycin, 2 mM L-glutamine, and 10% fetal
calf serum (Invitrogen). BMDC were obtained by extracting bone marrow
from the leg bone of Balb/c mice (Harlan UK, Blackthorn, UK). The cells
obtained were washed twice in sterile phosphate-buffered saline and
plated out onto tissue culture dishes. Cells were grown in complete
media supplemented with 5 ng/ml granulocyte
macrophage-colony-stimulating factor (30). Media was changed on
alternating days for 10 days. Cells were used in studies on day 10 of
primary culture.
CD40 Antibodies--
Rat monoclonal antibody 3/23 was raised
against mouse CD40 (31). Mouse anti-human CD40 monoclonal antibody
LOB7.6 was obtained from Serotec, UK.
Plasmid DNA--
All plasmid DNA was prepared using a commercial
DNA extraction and isolation kit (Maxiprep, Qiagen). IL-6 promoter
function was studied using the luciferase reporter vector pIL6-Luc651, containing nucleotides 
651 to +1 of the human IL-6 gene. Construction of pIL6-Luc651 and derivatives carrying site-directed mutations in the
AP-1 (283 to 276), NF-IL6 (154 to 146), and NF-B (72 to
63) sequences have been described elsewhere (26). A luciferase reporter vector containing nucleotides 332 to +35 of the human IB- gene promoter was provided by Professor Ron Hay (St. Andrews, UK). The control Renilla luciferase vector pRL-TK was purchased from
Promega (Southampton, UK). An expression vector (pRSV-JunD) for
dominant negative JunD lacking amino acids 1-162 has been previously
described (27). Expression vector pJH282 for CBF1 was obtained from Dr.
Diane Hayward (Baltimore, MD). Dominant negative TRAF2 was produced
from an expression vector obtained from Professor David Brenner (Chapel
Hill, NC) that generates a TRAF2 protein lacking nucleotides 87-501
which prevents interactions with downstream effector molecules (32).
Dominant negative TRAF6 was overexpressed from a vector provided by
Professor Luke O'Neill (33). An expression vector encoding an IB
super-repressor protein (IBD) has been described elsewhere (34).
Wild type human CD40 (hCD40) was amplified from Raji cell line cDNA
using h40HindIIIF 5'-ccaagcttcacctcgccatggttcgtctgc-3' sense and
h40XbaIR 5'-gtgggtctagactcactgtctctcctgcac-3' antisense primer which
incorporate HindIII and XbaI restriction enzyme
sites to allow for cloning of hCD40 into pcDNA3 expression vector.
Human CD40 expression constructs were generated by two-step recombinant
PCR using hCD40 wild type as template for hCD40KKV, hCD40T254A, and
hCD40T254S whereas both hCD40 and hCD22 (a gift of Professor Martin
Glennie, Southampton, UK) expression constructs were used as templates for generation of hCD40/hCD22 chimeric protein. The general method for
two-step recombinant PCR was as follows; first step PCR was constructed
as two separate reactions. Reaction 1 was set up using h40HindIIIF
primer and the appropriate antisense primer for the given mutant as
listed in Table I. In all reactions we
used 1 µg of hCD40wt/pcDNA3 template, 100 ng of
h40HindIIIF primer, and 100 ng of appropriate antisense primer, 2.5 µl of optimized Pfu polymerase buffer (Promega), 0.4 mM dNTP mixture, and 2 units of Pfu polymerase
in a total reaction volume of 25 µl. Reaction 2 was set up in the
same manner using h40XbaIR primer and the appropriate sense primer for
the given mutant as listed in Table I. In each case both the sense and
antisense primers carry the required mutation or a stop codon (as for
hCD40KKV). For all first step reactions hCD40wt/pcDNA3 was used as
template, except for reaction 2 of hCD40/hCD22 where hCD22 expression
construct was used instead. PCR was carried with an initial 5-min
incubation at 94 °C. This was followed by 30 cycles of a 1-min
annealing step at temperatures outlined in Table I, a 2-min elongation step at 72.0 °C, and a 45-s denaturation step at 94 °C. After 30 cycles a final elongation reaction was carried out for 10 min at
72.0 °C. PCR products were separated by electrophoresis at 80 V for
60 min through a 1% agarose gel and detected by ethidium bromide
staining. Expected sizes of specific PCR products (as in Table I) were
verified by reference to a 1-kilobase DNA ladder. PCR products were
then excised from agarose gels, purified, and eluted in 20 µl of
dH2O. The second step PCR reaction was set up
using 10 µl of each of the first step PCR reactions, 2.5 µl of
optimized Pfu polymerase buffer (Promega), 0.4 mM dNTP mixture, and 2 units of Pfu polymerase
in a total reaction volume of 25 µl. The PCR program was set up as
described for first step reactions, with annealing temperature as shown
in Table I for each mutant. A total number of 15 cycles was used,
followed by addition of h40HindIIIF and h40XbaIR end primers and
further 15 PCR cycles. The PCR products were digested with
HindIII and XbaI, separated on 1% agarose gel,
excised, purified, and ligated into pcDNA3. Presence of the
required mutation was confirmed by sequencing. Human CD40 expression
constructs hCD40T254E, hCD40T254A
262, and hCD40262 were a gift
from Professor Lawrence Young (Institute for Cancer Studies,
University of Birmingham, UK).
EMSA--
NF-B and AP-1 DNA binding was determined by EMSA as
previously described (27) using a 32P end-labeled
double-stranded oligonucleotide probe containing a consensus NF-B or
AP1 site as contained within the human IL-6 promoter sequence. NF-B
sense oligonucleotide was: 5'-caaatgtgggattttcccatga-3'; and
antisense oligonucleotide, 5'-tcatgggaaaatcccacatttg-3'; AP1 sense
oligonucleotide was 5'-aaagtgctgagtcactaataa-3', and antisense nucleotide 5'-ttattagtgactcagcacttt-3'. Nuclear extracts were prepared
from FSDCs by a protocol modified from that described by Dignam
et al. (35). Harvested cells were washed twice in ice-cold
phosphate-buffered saline prior to lysis in Buffer A supplemented with
0.2% Nonidet P-40, 0.5 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 0.2 mM EDTA, and 15 µg/ml aprotinin. Lysates
were centrifuged for 10 s at 13,000 rpm to collect crude nuclear
pellets. Supernatants were discarded and pellets were washed twice in
lysis buffer prior to resuspension in Buffer C supplemented with 0.5 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 0.2 mM EDTA, and 15 µg/ml aprotinin. After a 10-min incubation on ice with occasional vortexing, the extracts were cleared
of insoluble nuclear material by centrifugation at 13,000 rpm for
30 s. Cleared nuclear extracts were transferred to fresh Eppendorf
tubes, and their protein content was determined using the Bradford DC
assay kit (Bio-Rad). EMSA reactions were assembled on ice and consisted
of an initial 10-min incubation of 4 µl of Buffer C containing 2-10
µg of nuclear protein extract and 12 µl of water containing 2 µg
of poly(dI-dC). 4 µl of water containing 0.4 ng of radiolabeled
double-stranded AP-1 probe was then added to the reaction and, after
mixing, was incubated for a further 20 min. For supershift assays,
reactions were incubated for a further 16 h in the presence of 2 µg of anti p50/p65 or anti-Jun antiserum (Santa Cruz Biotechnology,
Inc.). EMSA and supershift reaction mixtures were then resolved by
electrophoresis on an 8% nondenaturing polyacrylamide gel
(37:5:1).
SDS-Polyacrylamide Gel Electrophoresis and
Immunoblotting--
Crude Nonidet P-40 soluble cytoplasmic extracts
were prepared by lysis of phosphate-buffered saline-washed cultures in
Dignam buffer A supplemented with 0.2% Nonidet P-40. Lysates were
centrifuged for 5 s at 13,000 rpm to collect nuclei. Supernatants
were transferred to fresh Eppendorf tubes and centrifuged for a further
1 min at 13,000 rpm to pellet Nonidet P-40 insoluble material,
supernatants were then designated as crude cytoplasmic extracts.
Nuclear extracts were prepared as described for EMSA reactions. 30 µg
of cytoplasmic or nuclear extracts from untreated FSDCs or treated with
30 µg/ml anti-mouse CD40 3/23 mAb were then fractionated by
electrophoresis through a 9% SDS-polyacrylamide gel. Gels were run at
a 100 V for 1.5 h prior to transfer onto nitrocellulose as
previously described (27). Following blockade of nonspecific protein
binding, nitrocellulose blots were incubated for 1 h with primary
antibodies (diluted in TBS/Tween 20 (0.075%)) containing 3% Marvel
and 3% bovine serum albumin. Rabbit polyclonal antibodies recognizing JunD, JunB, and c-Jun (Santa Cruz Biotechnology, Inc.) were used at a
final concentration of 1 µg/ml. Blots were then washed three times in
TBS/Tween 20 prior to incubation for 1 h with sheep anti-rabbit horseradish peroxidase antibody (1:2000). After extensive washing in
TBS/Tween 20 the blots were processed to distilled water for detection
of antigen using the ECL system (Amersham Biosciences, Buckinghamshire,
United Kingdom).
Reverse Transcriptase-Polymerase Chain Reaction
(RT-PCR)--
mRNA was extracted from 1 × 107
FSDC cells which were either left untreated or were treated with
30 µg/ml anti-mCD40 mAb 3/23 for 24, 48, and 72 h using the
"Quickprep micro mRNA Purification kit" (Amersham Biosciences)
as per the manufacturer's instructions. 5 µg of mRNA obtained
was used to generate first strand cDNA using random hexamer primer
(p(dN)6) and "First strand cDNA synthesis system"
(Amersham Biosciences) as per the manufacturer's instructions. PCR
amplification of mouse IL-6, IL-12 (p35 and p40), IL-4, IFN, TRAF2,
TRAF6, and 
-actin cDNAs was carried out using specific oligonucleotide primers selected within the coding regions of the
genes. IL-6 primers used were 5'-actgatgctggtgacaac- 3' (sense) and
5'-tccacaaactgatatgct-3' (antisense) designed to produce a 620-bp
product; IL12p35 primers were 5'-ataaccatgggtctcccaaggtca-3' (sense)
and 5'-gccttactagttcaggcggagctc-3' (antisense) designed to produce a
739-bp product; IL12p40 primers were 5'-agcaccatgggtcctcagaagcta-3' (sense) and 5'-cgtactagtctaggatcggaccctgca-3' (antisense) designed to
produce a 1007-bp product; IFN primers were
5'-ggccatggtctgagacaatgaacg-3' (sense) and
5'-cctctagagaatcagcagcgactc-3' (antisense) designed to produce a 488-bp
product; IL-4 primers were 5'-acagagctattgatgggtctcaacc-3' (sense) and
5'-tttccaggaagtctttcagtgatgt-3' (antisense) designed to produce a
400-bp product; TRAF2 primers were 5'-atgaaggcctgtatgaagaag-3' (sense)
and 5'-ttctcagtctccaccatctct-3' (antisense) designed to produce a
472-bp product; TRAF6 primers were 5'-gaaagatgacagcgtgagtgg-3' (sense)
and 5'-cacggacgcaaagcaaggtta-3' (antisense) designed to produce a
768-bp product; and -actin primers were 5'-tggaatcctgtggcatccat-3' (sense) and 5'-taaaacgcagctcagtaaca-3' (antisense) designed to produce
a 500-bp product. PCRs were composed of 1 µl of cDNA template, 100 ng each of sense and antisense oligonucleotide primers, 2.5 µl of
optimized TaqPCR buffer (Promega), 0.4 mM dNTP
mixture, and 2 units of Taq polymerase in a total reaction
volume of 25 µl. Following an initial 5-min incubation at 94 °C,
PCRs were performed using a 1-min annealing step (at 50 °C for IL-6,
59 °C for IL12p35, 57.5 °C for IL12p40, 54 °C for IL-10,
56 °C for IFN and IL-4, 55.7 °C for TRAF2, 55.4 °C for
TRAF6, and 55 °C for -actin), followed by a 2-min elongation step
at 72.0 °C and a 45-s denaturation step at 94 °C. A total number
of 27, 30, 30, 35, 35, 35, 35, and 27 PCR cycles were carried out for
detection of IL-6, IL12p35, IL12p40, IFN, IL-4, TRAF2, TRAF6, and
-actin, respectively, followed by a final elongation reaction for 10 min at 72.0 °C. PCR products were separated by electrophoresis at 80 V for 60 min through a 1% agarose gel and were detected by ethidium
bromide staining. Expected sizes of specific PCR products (620, 739, 1007, 488, 400, 472, 768, and 500 bp for IL-6, IL12p35, IL12p40,
IFN, IL-4, TRAF2, TRAF6, and -actin, respectively) were verified
by reference to a 1-kilobase DNA ladder.
Transfections and Reporter Gene Assays--
FSDCs and DC2.4s
were transfected by the non-liposomal Effectene protocol (Qiagen)
according to the manufacturer's instructions. Luciferase assays were
performed using a dual luciferase kit (Promega) according to the
manufacturer's instructions. IL-6 promoter-driven expression of
firefly luciferase was normalized for differences in transfection
efficiency by measurement of the activity of a co-transfected Renilla
luciferase vector (pRLTK).
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RESULTS |
CD40-stimulated Induction of Cytokine mRNAs in Primary Immature
Murine Bone Marrow-derived DCs and FSDCs--
To circumvent problems
associated with heterogeneity of isolated primary DCs and to generate
sufficient cell numbers to perform an in depth analysis of CD40
regulation of IL-6 gene transcription in DCs we used an established
cell line. FSDC were originally generated by retroviral-mediated
immortalization of DC progenitors from fetal mouse skin and resemble
immature BMDC in terms of their morphology, function, and antigenic
phenotype (28). Prior to use of FSDC for gene transcription studies we
established by fluorescence-activated cell sorter analysis that FSDC
express surface CD40 (data not shown). We then determined the relative
ability of primary immature murine BMDC and FSDC to express a variety
of cytokine transcripts in response to stimulation for 24 h with
an anti-mouse CD40 mAb (3/23). Fig. 1
shows RT-PCR analysis of IL-6, IL-12 (p35 and p40), IFN, and IL-4 in
unstimulated () and anti-CD40 stimulated (+) BMDC or FSDC. The data
show a similar profile for cytokine mRNA expression in BMDC and
FSDC with both cells undergoing a strong induction of IL-6 and both the
p35 and p40 subunits of IL-12, by contrast neither of the two cell
cultures displayed expression of IFN or IL-4. These data suggest
that FSDC are a good model for studying the transcriptional induction
of IL-6 gene expression by engagement of CD40.
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Fig. 1.
Induction of cytokines in FSDCs and BMDCs
during culture with anti-CD40 mAb 3/23. mRNA was isolated from
FSDCs or BMDCs which were either incubated with 30 µg/ml anti-CD40
mAb 3/23 or left untreated for 24 h. The mRNA was used to
obtain first strand cDNA, which was used as a template in RT-PCR
reactions using protocols described under "Materials and Methods."
Utilizing this method, cDNA species encoding murine IL-6, IL-12
(p35 and p40), IFN, IL-4, and -actin were amplified. For IFN
and IL-4, positive control RT-PCR reactions are shown on the far
left-hand side and used mRNA extracted from total blood
lymphocytes (TBL) taken from a Balb/c mouse. The gels shown
are representative of at least two independent experiments. A
1-kilobase DNA ladder was run alongside the PCR products to confirm
correct sizes of the amplified cDNA fragments.
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Requirement for AP1, NF-B, and NF-IL-6 Sites for CD40 Induction
of IL-6 Promoter Activity in FSDC--
FSDC were transfected with wild
type and mutant IL-6-promoter-luciferase constructs together with a
control TK-renilla-luciferase vector to normalize data for differences
in transfection efficiency. The mutant IL-6 promoter constructs lacked
binding sites for AP1, NF-B, NF-IL-6, or NF-B + NF-IL-6 and have
recently been described elsewhere (26, 27). The IL-6 promoter had a
weak basal level of transcription that was induced by 20.5-fold in
cells stimulated for 24 h with anti-CD40 (fig
2, A and B). These
basal and inducible promoter activities therefore reflect the IL-6
mRNA expression data presented in Fig. 1. Mutation of the NF-B
site in the IL-6 promoter reduced both basal and CD40-induced
transcription such that fold induction in response to anti-CD40 was
just 6.4-fold (Fig. 2, A and B). Disruption of
NF-IL-6 binding to the promoter mainly affected basal transcription,
with the induction in response to CD40 at 24.5-fold being higher than
for the wild type promoter. However, a double mutation that perturbed
both NF-B and NF-IL-6 binding almost completely abolished the
ability of anti-CD40 to induce promoter activity (1.5-fold). These data
indicate that NF-B and NF-IL-6 may act in synergy to regulate CD40
signaling at the IL-6 promoter. Mutagenesis of the AP1 site in the IL-6 promoter had a minor effect on basal transcription but reduced anti-CD40-inducible promoter activity to 11-fold, which is almost a
50% reduction of transcription relative to the wild type promoter. Induction of high level IL-6 gene transcription in FSDC by engagement of surface CD40 therefore requires all three transcription factor binding motifs in the IL-6 promoter.
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Fig. 2.
Induction of IL-6 promoter activity by
anti-CD40 and requirement for specific regulatory DNA motifs.
A, FSDCs were transfected with 100 ng of pRLTK and 1 µg of
either wild type pIL6-Luc651 or mutated pIL6-Luc651 constructs carrying
mutation in the AP1, NF-B, and NF-IL6 sites. The transfected
cultures were split into two flasks. 24 h later, one culture flask
was incubated with 30 µg/ml anti-CD40 mAb 3/23 for a further 24 h, while the remaining flask was left untreated. Samples treated with
3/23 are depicted in black solid boxes, whereas untreated
samples are in white. Luciferase activities were normalized
to pRLTK activity and expressed as the mean ± S.E. of three
independent transfection experiments. Statistical analysis was
performed by Student's t test. *, **, and *** denote
p < 0.05, 0.01, and 0.005, respectively. B,
fold induction of the sample incubated with anti-CD40 mAb 3/23
versus the non-treated sample for each of the pIL6-Luc651
constructs.
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Induction of NF-B by Anti-CD40--
To confirm a role for
NF-B in the regulation of CD40-inducible IL-6 gene transcription,
FSDC were co-transfected with the wild type IL-6 promoter construct and
an expression vector for an IB- super-repressor protein (IBD)
that lacks Ser32 and Ser36 residues required
for IKK-mediated phosphorylation and subsequent proteasome-mediated
degradation (34). Overexpression of this mutated IB- therefore
leads to capture of NF-B dimers in irreversibly inactive complexes.
FSDC overexpressing the IBD displayed a reduction in both the basal
and anti-CD40 induced IL-6 promoter activities relative to cells
co-transfected with the control empty vector (fig
3A). When fold induction by
anti-CD40 was determined we found that overexpression of IBD caused
a diminution of inducible IL-6 promoter activity from 21.6-fold
(control) to 7.7-fold (Fig. 3B). This level of inhibition
was similar to the drop in fold-induction observed when the NF-B
site of the IL-6 promoter was disrupted (Fig. 2B).
Activation and DNA binding of NF-B are therefore required for
induction of IL-6 gene transcription in CD40-stimulated FSDC. To
determine whether there is a similar requirement in primary DCs we
determined the effects of the NF-B inhibitor MG132 (36) on CD40
induction of IL-6 mRNA expression in BDMC. As shown in the
representative gel in Fig. 3C inhibition of NF-B
activation by MG132 completely blocked CD40 induced elevation of IL-6
transcript.
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Fig. 3.
Induction of the IL-6 promoter requires
activation of NF-B. A, FSDCs were
transfected with 100 ng of pRLTK, 1 µg of wild type pIL6-Luc651, and
2 µg of IB trans-dominant negative (IBD) expression vector
or pcDNA3 as control. The transfected cultures were split into two
flasks. 24 h after transfection, one flask was incubated with 30 µg/ml anti-CD40 mAb 3/23 for 24 h while the remaining flask was
left untreated. Luciferase activity was determined 48 h after
transfection. Luciferase activities were normalized to pRLTK activity
and expressed as the mean ± S.E. of three independent
transfection experiments. Samples treated with 3/23 are depicted in
black solid boxes, whereas untreated samples are in
white. Statistical analysis was performed by Student's
t test. *** denotes p < 0.005. B, fold induction of the samples incubated with anti-CD40
mAb 3/23 versus the non-treated samples as shown in Fig.
3A. C, mRNA was isolated from BMDCs which
were either untreated () or incubated for 24 h with 30 µg/ml
anti-CD40 mAb 3/23 together with either 5 µM MG132
dissolved in Me2SO or Me2SO alone. The mRNA
was used to obtain first strand cDNA, which was used as a template
in RT-PCR. cDNA species encoding murine IL-6 and -actin were
amplified over 32 and 27 cycles in RT-PCR reactions using protocols
described under "Materials and Methods." The gels shown are
representative of at least two independent experiments. A 1-kilobase
DNA ladder was run alongside the PCR products to confirm correct sizes
of the amplified cDNA fragments.
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We next determined if anti-CD40 treatment of FSDC induced NF-B DNA
binding activities using a double stranded oligonucleotide carrying the
NF-B binding sequence from the IL-6 promoter as an EMSA probe (fig
4A). Unstimulated FSDC
(lanes 3, 5, 7, 9, and 11) expressed
two NF-B DNA binding complexes, a weak low mobility complex (complex
2) and a more intense higher mobility interaction (complex 1). Both
interactions were found to be sequence specific (data not shown) and
were induced following a 24-h exposure of FSDC to anti-CD40 (compare
lanes 2 and 3), however, the induction of complex
2 was more impressive than complex 1. For reasons not yet known the
mobility of complex 1 was occasionally slightly altered in
CD40-stimulated cells. To determine the protein components of the two
CD40-inducible NF-B complexes we performed supershift/antibody interference experiments. Complex 2 was reactive with both anti-p50 (lane 4) and anti-p65 (lane 6) antisera, moreover
incubation of pre-formed protein-DNA complexes with a combination of
the two antibodies (lane 8) resulted in almost total
interference of complex 2. By contrast, complex 1 was not reactive with
either the p50 or p65 antisera either alone or in combination. From
these data we conclude that the major inducible NF-B DNA binding
activity of anti-CD40 stimulated FSDC is the classic p50:p65
heterodimer. Previous studies have shown that the NF-B DNA-binding
site in the IL-6 promoter overlaps with a sequence that acts as a
binding site for the transcriptional repressor CBF1 (also designated
RBP-J) (37, 38). Since this overlapping sequence was included in our
NF-B EMSA probe, we therefore investigated the possibility that
NF-B complex 1 may be due to binding of CBF1. As precise nucleotides
required for binding of p50:p65 and CBF1 have previously been
identified we carried out EMSA using double stranded oligonucleotide probes carrying point mutations that disrupt binding of either p50:p65
or CBF1 (Fig. 4B). Loss of a nucleotide required for p50:p65 binding resulted in disrupted assembly of complex 2 but had no effect
on complex 1, this confirms that p50:p65 heterodimers are the protein
components of complex 2. In contrast, point mutations that prevent CBF1
binding had no effect on assembly of complex 2 but resulted in a loss
of complex 1 formation. Confirmation that CBF1 was the
protein component of complex 1 was obtained from supershift/antibody
interference experiments in which we showed that an antisera raised
against CBF1 could specifically interfere with complex 1 (Fig.
4C).
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Fig. 4.
EMSA analysis of
NF-B and CBF1 in anti-CD40 stimulated FSDC.
A, nuclear extracts from control or FSDC cells treated with
anti-mCD40 mAb 3/23 for 24 h were isolated and 2 µg used in EMSA
with NF-B double stranded oligonucleotide probe. Two (1 and 2)
specific DNA-protein complexes were assembled and are denoted by
arrows. Supershift analysis was performed on control and
treated samples using antisera recognizing p50 and p65 or JunB as a
control. B, 2 µg of nuclear extracts from FSDC cells
treated with anti-mCD40 mAb 3/23 for 24 h (as obtained in
A) were incubated with double stranded NF-B
oligonucleotide probe, or NF-B probe lacking p50/p65 or CBF1-binding
sites. The nucleotide substitutions introduced into the wild type
NF-B oligonucleotide to generate mutant oligonucleotides lacking
p50/p65 or CBF1-binding sites are shown below the EMSA gel.
C, 2 µg of nuclear extracts from FSDC cells treated with
anti-mCD40 mAb 3/23 for 24 h (as obtained in A) were
incubated with double stranded NF-B oligonucleotide probe and
supershift analysis performed using antisera recognizing p50, p65,
CBF1, or JunB as a control.
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AP1 Is Induced by Anti-CD40 and Is Required for Stimulation of IL-6
Promoter Activity--
As mutagenesis of the AP1 site of the IL-6
promoter resulted in roughly a 50% drop in anti-CD40 stimulated
transcription (Fig. 2, A and B) it was of
interest to determine whether engagement of surface CD40 induces AP1
DNA binding activity in FSDC. EMSA analysis of protein-DNA complex
formation was carried out using a double stranded oligonucleotide probe
carrying the AP1-binding site from the IL-6 promoter. Fig.
5A shows that unstimulated
FSDC express a weak AP1 DNA binding activity that was strongly induced in cultures treated for 16 h with anti-CD40. Ability of a 100-fold excess of unlabeled AP1 sites to compete for binding and lack of
competition by a 100-fold excess of nonspecific (SP1 site) double
stranded oligonucleotides confirmed that the AP1 protein-DNA complexes
in FSDC were specific and saturable (Fig. 5A). As
transcriptionally active AP1 complexes must contain a Jun (c-Jun, JunB,
or JunD) factor either in homodimeric (Jun:Jun) or heterodimeric (Jun
in partnership with a Fos family protein) forms, we used
supershift/antibody interference to identify the Jun components of the
CD40-inducible AP1 DNA binding activity of FSDC. As shown in Fig.
5B antisera recognizing c-Jun, JunB, and JunD reduced the
intensity of AP1 DNA binding activity indicating that all three
proteins may be present in the complex. However, JunB and JunD antisera
not only reduced complex formation to a higher degree than the c-Jun
antisera, but they also promoted the assembly of readily detectable
supershift complexes. Hence JunB and JunD are likely to be the
predominant Jun components of the induced AP1 complexes. We were also
able to show that an antisera raised against c-Fos interfered with AP1
complex formation, hence anti-CD40 treatment may induce both Jun:Jun
and Jun:Fos AP1 dimers in FSDC. Lack of supershift or interference in
EMSA reactions incubated with anti-SP1 antisera confirmed specificity
of the results obtained using the Jun and Fos antisera.
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Fig. 5.
Induction of AP-1 activity by CD40 ligation.
A, nuclear extracts from control or FSDC cells treated with
anti-mCD40 mAb 3/23 for 16 h were isolated and 10 µg used in
EMSA with AP1 double stranded oligonucleotide probe in presence of 100 M excess of unlabeled nonspecific oligonucleotide (Sp1) or
unlabeled specific AP1 oligonucleotide. B, supershift
analysis was performed on 3/23 mAb treated FSDC samples using antisera
recognizing c-Jun, JunD, JunB, c-Fos, or Sp1 as a control. Supershift
complexes are shown for extracts incubated with JunB and JunD antisera.
Asterisks placed to the left of the supershift
complexes are included to aid identification of these species.
C, immunoblot analysis of JunD, JunB, and c-Jun protein
expression was performed on crude cytoplasmic and nuclear extracts from
control and FSDCs treated with 3/23 mAb for 16 h. All gels are
representative of three independent experiments. D, FSDCs
were transfected with 100 ng of pRLTK, 1 µg of wild type pIL6-Luc651,
and 2 µg of JunD dominant negative expression vector pRSV-JunD or
pRSV as control. The transfected cultures were split into two flasks
and after 24 h one flask was incubated with 30 µg/ml anti-CD40
mAb 3/23 while the remaining flask was left untreated. Luciferase
activity was determined 48 h after transfection. Luciferase
activities were normalized to pRLTK activity and expressed as the
mean ± S.E. of three independent transfection experiments.
Samples treated with 3/23 are depicted in black solid boxes,
whereas untreated samples are in white. Statistical analysis
was performed by Student's t test. ** denotes
p < 0.01.
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As changes in AP1 activity can be regulated by either transcriptional
or post-translational events it was of interest to establish if
anti-CD40 treatment of FSDC alters the expression of Jun proteins. Fig.
5C shows a Western blot analysis of Jun protein expression in the cytoplasmic and nuclear fractions of unstimulated () and anti-CD40 stimulated (+) FSDC. All three Jun factors were expressed in
both the cytoplasm and nucleus of FSDC. The cytoplasmic pool of the Jun
factors was unchanged upon stimulation with anti-CD40, by contrast
nuclear levels of c-Jun were slightly elevated while nuclear JunB
expression was strongly induced. Confirmation of the requirement for
Jun activity in the IL-6 promoter response to anti-CD40 treatment was
obtained by co-transfection of FSDC with the IL-6 promoter and an
expression vector (RSV-JunD) for a dominant negative JunD protein.
The mutant JunD protein expressed from this vector lacks a functional
transactivation domain (27). As the different Jun proteins are able to
interact with each other to form functional AP1 binding dimers,
overexpression of the dominant negative JunD will sequester c-Jun,
JunB, and JunD proteins into functionally inactive dimers. Fig.
5D shows that co-transfection of RSV-JunD was without effect
on basal levels of IL-6 promoter activity but profoundly inhibited
CD40-induced transcription.
Structural Requirements in the Cytoplasmic Domain of CD40 Required
for Induction of IL-6 Promoter Activity--
Previous studies in B and
T cell lines have determined specific amino acid sequences in the
cytoplasmic tail of CD40 that are required for activation of
intracellular signaling in response to engagement of the receptor (2,
14-21). To determine the structural features of the CD40 cytoplasmic
domain required for induction of IL-6 gene transcription in FSDC we
employed the following strategy. A panel of wild type and mutant
(carrying deletions or point mutations in the cytoplasmic domain) human
CD40 expression vectors were co-transfected into FSDC with the IL-6
promoter-luciferase vector. The transfected culture was then exposed
for 24 h to LOB7.6, an anti-CD40 monoclonal antibody that
specifically recognizes human CD40 (hCD40) and lacks species
cross-reactivity. Fluorescence-activated cell sorter detection of
transfected hCD40 was used to confirm that the different expression
vectors generated quantitatively similar levels of immunoreactive cell
surface hCD40 (data not shown). FSDC transfected with wild type hCD40
displayed a 5.3-fold induction of IL-6 promoter activity in response to
LOB7.6 (Fig. 6A). However, in
contrast cells either transfected with the promoter alone or with
mutant CD40 expression vectors producing proteins that either lacked a
cytoplasmic tail (hCD40KKV) or had the cytoplasmic domain from CD22 in
place of the CD40 cytoplasmic domain were unable to induce IL-6
transcription in response to LOB7.6. These data show, as expected, that
the cytoplasmic domain of CD40 is critical for signaling. Mutation of
threonine 254 to a residue that is not a target for phosphorylation
(hCD40T254E and hCD40T254A) dramatically reduced LOB7.6 stimulation of
IL-6 promoter activity. However, hCD40 carrying a serine substitution
at this residue retained an ability to induce IL-6 promoter activity in
response to LOB7.6. Phosphorylated threonine 254 has been reported to
be critical for recruitment of TRAF2, -3, and -5 in response to
engagement of CD40 (15-18), our data therefore suggest a role for
these adaptor proteins in the induction of IL-6 gene transcription.
Construct hCD40262 lacks 15 amino acids (262-277) at the
COOH-terminal end of hCD40, which includes critical binding sites for
TRAF3 (Q263), TRAF5, and Jak3 but is not critical for TRAF2 binding (15, 16, 18, 39). This construct was able to support LOB7.6-induced IL-6 promoter activity albeit at a reduced level (60%) relative to the
induction obtained with wild type hCD40. A construct in which both
threonine 254 is mutated and the final 15 amino acids are deleted
(hCD40T254A262) was as expected unable to support LOB7.6 induction
of IL-6 gene transcription. These data suggest that neither TRAF3,
TRAF5, nor Jak3 play an essential role in anti-CD40 stimulation of IL-6
gene transcription in FSDC and indicate that TRAF2 is likely to be a
major signal transducer of this response.
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Fig. 6.
Requirement for specific amino acid sequences
in the cytoplasmic domain of CD40. A, 1 µg of wild type
pIL6-651Luc was transfected into FSDC cells along with 100 ng of pRLTK
and 2 µg of empty vector pcDNA3 or pcDNA3-derived expression
vectors carrying cDNA cassettes for wild type hCD40, truncated
hCD40 containing three remaining intracellular residues (hCD40KKV),
hCD40 mutants carrying point mutations (hCD40T254A, hCD40T254E, and
hCD40T254S), hCD40 with a point mutation and a deletion of
carboxyl-terminal 15 amino acids (hCD40T254A262), hCD40 with 15 carboxyl-terminal amino acids deleted (hCD40262), or a fusion
protein made up of hCD450 extracellular and transmembrane domains and
hCD22 intracellular domain. The transfected cultures were split into
two flasks, one flask was treated with 300 µg/ml anti-human CD40 mAb
LOB7.6 for 24 h and the other was left untreated. 24 h later,
cells were harvested and luciferase assays performed. Samples treated
with LOB7.6 are depicted in black solid boxes, whereas
untreated samples are in white. Luciferase activities were
normalized to pRLTK activity and expressed as the mean ± S.E. of
six independent transfection experiments. B, FSDC cells were
transfected with 1 µg of IB-Luc, 100 ng of pRLTK and hCD40
expression vectors and treated as already described in Fig.
5A. Luciferase activities were normalized to pRLTK activity
and expressed as the means ± S.E. of five independent
transfection experiments.
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Lee and colleagues (16) recently reported that TRAF2 mediates the
induction of ICAM-1 in CD40 stimulated B cells via activation of
NF-B and Jun-N-terminal kinase 1. To determine whether TRAF2 binding
is also required for induction of NF-B by engagement of hCD40 in
FSDC we co-transfected the panel of hCD40 expression vectors together
with an IB- promoter-luciferase reporter construct. Activity of
the IB- promoter is critically dependent on binding of NF-B,
forming a negative feedback mechanism for NF-B activation in cells
(40). Measurement of IB- promoter activity can therefore be used
as a sensitive and specific monitor of NF-B activation. Overexpression of wild type hCD40, hCD40T254S, and hCD40262 gave rise to an elevation of basal IB- promoter activity, all three constructs also supported a strong induction of promoter activity in
cells stimulated for 24 h with LOB7.6 (Fig. 6B). By
contrast, FSDC transfected with the remaining mutant hCD40 constructs
that disrupt TRAF2 binding in addition to TRAF3, TRAF5, and Jak3
displayed low basal levels of IB- promoter activity that were not
inducible by LOB7.6.
Inhibition of TRAF2 Signaling Blocks CD40 Activation of IL-6 Gene
Transcription--
RT-PCR was used to confirm that FSDC express TRAF2
and TRAF6 transcripts, with no detectable change in expression
following anti-CD40 treatment (fig
7A). To help confirm a major
role for TRAF2 in anti-CD40 stimulation of IL-6 gene transcription we
determined the effects of overexpressing a dominant negative TRAF2
protein on IL-6 promoter function. Co-transfection of the IL-6
promoter-luciferase reporter construct with an expression vector for a
dominant negative TRAF2 resulted in a complete inhibition of anti-CD40
stimulated transcription (Fig. 7B). By contrast
overexpression of a dominant negative TRAF6 protein had only a minor
effect on the responsiveness of the IL-6 promoter to anti-CD40
treatment (Fig 7C). We were also able to show that dominant
negative TRAF2 blocked anti-CD40 induction of IB- promoter
activity while again dominant TRAF6 had only a minor inhibitory effect
(data not shown). These data indicate a critical requirement for TRAF2
mediated signaling events as part of the pathways that stimulate
NF-B activity and IL-6 gene transcription in response to anti-CD40
treatment of FSDC.
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Fig. 7.
Inhibition of anti-CD40 induced IL-6 promoter
activity by dominant negative TRAF2. A, mRNA was
isolated from FSDCs which were either incubated with 30 µg/ml
anti-CD40 mAb 3/23, or left untreated for 24 h. The mRNA was
used to obtain first strand cDNA, which was used as a template in
RT-PCR reactions using protocols described under "Materials and
Methods." Utilizing this method, cDNA species encoding murine
TRAF2, TRAF6, and -actin were amplified over 35, 35, and 28 PCR
cycles, respectively. The gels shown are representative of at least two
independent experiments. A 1-kilobase DNA ladder was run alongside the
PCR products to confirm correct sizes of the amplified cDNA
fragments. B, FSDCs were transfected with 100 ng of pRLTK, 1 µg of IB-Luc, and 2 µg of TRAF2 dominant negative
(TRAF2 DN) expression vector or empty control vector. The
cultures were split into 2 and 24 h after the transfection
one-half was incubated with 30 µg/ml anti-CD40 mAb 3/23 for 24 h. Luciferase activity was determined 48 h after transfection.
C, FSDCs were transfected with 100 ng of pRLTK, 1 µg of
IB-Luc, and 2 µg of TRAF6 dominant negative (TRAF6 DN) and
processed as described for B. For both B and
C, luciferase activities were normalized to pRLTK activity
and expressed as the mean ± S.E. of three independent
transfection experiments. Samples treated with 3/23 are depicted in
black solid boxes, whereas untreated samples are in
white. Statistical analysis was performed by Student's
t test. *** denotes p < 0.005.
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Prolonged Anti-CD40 Stimulation of FSDC Leads to Reduced Basal
Expression of IL-6--
In the experiments we have described so far,
FSDC were stimulated with anti-CD40 for only 24 h. Since it is
possible that DCs may interact with CD40L bearing T cells for longer
periods of time it was important to investigate the longevity of the
IL-6 response. Fig. 8 shows an RT-PCR
analysis of IL-6 mRNA expression in FSDC incubated for 24, 48, and
72 h in the absence and presence of the anti-mouse CD40 monoclonal
antibody 3/23. As expected a 24-h stimulation of FSDC with 3/23 gave
rise to a strong induction of IL-6 mRNA expression. However, this
induction was terminated by 48 h and moreover was accompanied by
an almost complete loss of IL-6 transcript relative to its basal level
of expression detected in control unstimulated cells. After 72 h
stimulation there was a slight but incomplete recovery of basal IL-6
mRNA expression. We conclude that sustained engagement of surface
CD40 leads to a repression of IL-6 gene transcription. To establish a
mechanism for this response we used EMSA to determine the
time-dependent effects of anti-CD40 stimulation on NF-B
DNA binding activities (Fig.
9A). Maximal induction of the
p50·p65 complex was observed at 16 h of stimulation after which
there was a modest but steady decline in the intensity of this complex.
By contrast the CBF1 protein-DNA interaction remained at an elevated
level after 48 h of stimulation. The effects of increased CBF1
expression on IL-6 promoter activity was tested by co-transfecting FSDC
with the IL-6 promoter-luciferase reporter and an expression vector for
CBF1. As shown in Fig. 9B, overexpression of CBF1
dramatically repressed both basal and anti-CD40 induced IL-6 promoter
activity. We therefore suggest that decline in activation of p50:p65
NF-B coupled with prolonged elevation of CBF1 DNA binding activity may contribute to the reduced basal expression of IL-6 in FSDC stimulated with anti-CD40 for periods in excess of 24 h.
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Fig. 8.
Induction of IL-6 gene transcription by
anti-CD40 is a transient response. mRNA was isolated from
control or FSDCs treated with anti-mCD40 mAb 3/23 for 24, 48, or
72 h. mRNA was used to obtain first strand cDNA which was
used as a template in RT-PCR reactions using protocols described under
"Materials and Methods." Utilizing this method, cDNA species
encoding murine IL-6 and -actin were amplified. The gels shown are
representative of at least two independent experiments. A 1-kilobase
DNA ladder was run alongside the PCR products to confirm correct sizes
of the amplified cDNA fragments.
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Fig. 9.
Prolonged induction of CBF1 DNA binding
activity and repression of IL-6 promoter function. A, 2 µg
of nuclear extract isolated from FSDC cells treated with anti-mCD40 mAb
3/23 for 0, 2, 4, 6, 16, 24, and 48 h was used in EMSA with
NF-B double stranded oligonucleotide probe. B, FSDCs were
transfected with 100 ng of pRLTK, 1 µg of wild type pIL6-Luc651, and
2 µg of CBF1 expression vector pJH282 or empty vector pSG5. The
cultures were split into two flasks and 24 h after transfection
one flask was incubated with 30 µg/ml anti-CD40 mAb 3/23 for 24 h, while the remaining flask was left untreated. The cells were then
harvested and luciferase assay performed. Samples treated with 3/23 are
depicted in black solid boxes, whereas untreated samples are
in white. Luciferase activities were normalized to pRLTK
activity and expressed as the mean ± S.E. of three independent
transfection experiments. Statistical analysis was performed by
Student's t test. *** denotes p < 0.005.
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Signaling Events Downstream of CD40 Are Conserved in DC2.4
and FSDC Cell Lines--
Having determined the intracellular signaling
events involved in the induction of IL-6 gene transcription in
3/23-stimulated FSDC it was considered important to verify our findings
in a second mouse bone marrow-derived DC cell line DC2.4. RT-PCR
analysis of IL-6 expression revealed that although DC2.4 expressed a
higher basal level of IL-6 mRNA than FSDC there was a significant
induction of expression at 24 h of anti-CD40 stimulation that with
further stimulation was reduced to below basal levels as observed with FSDC (data not shown). We then determined the role played by NF-B (Fig. 10A), AP1 (Fig.
10B), TRAF2 (Fig. 10C), TRAF6 (Fig.
10D), and CBF1 (Fig. 10E) in DC2.4. Treatment of
DC2.4 with 3/23 for 24 h resulted in a reproducible 2-3-fold
induction of IL-6 promoter activity, which is a weaker response than
observed for FSDC. However, the effects of attenuating each of the five
signaling molecules were similar between DC2.4 and FSDC. Inhibition of
TRAF2 and NF-B resulted in a total inhibition of 3/23 induced
transcription, by contrast expression of dominant negative TRAF6 had
only a minor inhibitory effect. Expression of dominant negative JunD
significantly reduced 3/23 induced transcription although this effect
was not as pronounced as that observed in FSDC. Finally overexpression of CBF1 dramatically repressed basal and anti-CD40 inducible IL-6 promoter activity in DC2.4. These data therefore support the generality of our findings in FSDC for mouse DC lines.
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Fig. 10.
Conservation of CD40 signaling events
between two distinct mouse DC lines (FSDC and DC2.4). To confirm
the generality of findings with FSDC a series of key experiments were
repeated using the mouse DC line DC2.4. DC2.4s were transfected with
100 ng of pRLTK, 1 µg of wild type pIL6-Luc651wt; A, 2 µg of IB dominant negative (IB DN) expression vector or
pcDNA3 empty vector as a control; B, 2 µg of JunD
dominant negative (pRSV JunD) expression vector or pRSV empty vector
as a control; C, 2 µg of TRAF2 dominant negative (TRAF2
DN) expression vector or pRK5 empty vector as a control; D,
2 µg of TRAF6 dominant negative (TRAF6 DN) expression vector or
pcDNA3 empty vector as a control; and E, 2 µg of CBF1
dominant negative (pJH282) expression vector or pSG5 empty vector as a
control. The transfected cultures were split into two flasks. 24 h
after transfection, one flask was incubated with 30 µg/ml anti-CD40
mAb 3/23 for 24 h while the remaining flask was left untreated.
Luciferase activity was determined 48 h after transfection.
Luciferase activities were normalized to pRLTK activity and expressed
as the mean ± S.E. of three independent transfection experiments.
Samples treated with 3/23 are depicted in solid black boxes,
whereas untreated samples are in white. Statistical analysis
was performed by Student's t test. * and *** denote
p < 0.05 and p < 0.005, respectively.
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DISCUSSION |
Despite the emergence of DCs as key cellular players
in the immune system, the signaling events that regulate DC function are poorly understood. In the present study we have made a detailed analysis of the molecular events that lead to transcriptional induction
of IL-6 in DCs stimulated via the CD40 signaling pathway. We have shown
that engagement of CD40 on DCs leads to a powerful, but transient
induction of IL-6 mRNA expression. This induction was maximal at
24 h after addition of anti-CD40 to cultures and was followed by a
rapid diminution of IL-6 transcript to levels that were less than those
found in unstimulated cultures. By investigating the structural
requirements at both the CD40 cytoplasmic tail and the IL-6 promoter,
together with establishing changes in the activity of specific
transcription factors, we have been able to determine the mechanisms
underlying these CD40 stimulated responses in DCs.
The IL-6 promoter was weakly active in FSDC, as expected from the low
but detectable level of IL-6 transcript found in both FSDC and primary
BMDC. Anti-mouse CD40 mAb 3/23 stimulated a powerful induction of IL-6
promoter activity in FSDC, as did anti-human CD40 mAb when added to
FSDC transfected with a human CD40 expression construct. These
observations contradict an earlier study using the B cell line CH12.LX
in which it was shown that while membrane-bound CD40L could induce IL-6
promoter activity and mRNA expression, anti-CD40 mAb could not
(14). Another discrepancy between this latter study and ours is the
role of NF-B, the transcription factor was apparently dispensable
for CD40 activation of IL-6 induction in CH12.LX (14), yet was critical
for CD40-induction of IL-6 promoter function in FSDC, DC2.4, and
primary bone marrow-derived DCs. The precise reasons for these
discrepancies are not known, however, it is possible that there are
inherent differences in either the phenotype of the cell lines or the
quality of the antibodies used in the two studies. Whatever these
differences may be, our study clearly demonstrates that engagement of
endogenous mouse CD40 or transfected human CD40 by specific antibodies
can induce IL-6 promoter activity in FSDC. This induction was dependent
on the presence of intact DNA-binding sites for NF-B and AP-1. Both basal and anti-CD40 inducible activities of the IL-6 promoter were
regulated by NF-B. Furthermore, we were able to show that blockade
of the activation of NF-B by the proteasome inhibitor MG132 resulted
in a total inhibition of anti-CD40 induced IL-6 mRNA expression in
primary BMDC. By contrast, AP-1 appeared to be mainly required for
inducible IL-6 promoter activity, since mutation of the AP-1 site of
the IL-6 promoter or overexpression of a dominant negative JunD protein
only had a significant negative effect on the promoter in cells treated
with anti-CD40.
Engagement of endogenous CD40 on FSDC by addition of mAb 3/23 resulted
in a transient induction of AP-1 DNA binding activity that was maximal
after 16 h of stimulation. A single, but diffuse AP-1 DNA-protein
complex was induced by 3/23 and consisted of all three Jun proteins
together with at least one Fos family protein, c-Fos. Examination of
Jun protein expression revealed that the major alteration in 3/23
stimulated cells was elevated nuclear expression of JunB. Although we
did not detect any significant changes in the expression of c-Jun or
JunD, the presence of both these proteins in the nucleus of FSDC,
together with our ability to detect them in the anti-CD40-induced AP-1
complex indicates that post-translational events probably regulate
their activity in FSDC. Of relevance to this idea, it is well
established that engagement of CD40 results in the activation of the
Jun N-terminal kinases (JNKs), which in turn are able to attenuate
c-Jun and JunD activities (2). Previous studies in B cells have shown that engagement of CD40 induces c-Jun, JunB, JunD, c-Fos, FosB, and
Fra1 at either the mRNA or protein level (41-43). However, few
studies have investigated CD40 mediated induction of AP-1 activity or
Jun and Fos protein family expression in monocytes or DCs. Revy
et al. (19) suggested that CD40 stimulation of human
monocytes was associated with induction of NF-B but not AP-1. Hence
it is possible that monocytes and DCs differ in their ability to
activate AP-1 in response to engagement of CD40, however, confirmation
of this idea requires detailed comparative studies that are beyond the
scope of the present study. From our data we can conclude that CD40
signaling in FSDC leads to the activation of a variety of Jun (c-Jun,
JunB, and JunD) containing AP-1 dimers. We can also conclude that these
AP-1 dimers are transcriptionally active and in combination with
NF-B and NF-IL-6 are able to stimulate the elevated levels of IL-6
gene transcription observed in anti-CD40 stimulated FSDC.
Activation of NF-B is a widely documented consequence of CD40
signaling and is known to be required for many CD40-induced changes in
gene expression (2). There is also a wide literature concerning the
role of TRAFs as mediators of CD40-induced NF-B activation, however,
there is controversy regarding the requirement for specific TRAFs in
this response (14-16, 18). Studies in 293 cells have shown that
NF-B and JNK can be activated by overexpression of TRAF2, TRAF5, and
TRAF6, by contrast TRAF1, TRAF3, and TRAF4 are unable to activate
either NF-B or JNK when overexpressed (16). Our data suggest that
TRAF2 is the critical mediator of anti-CD40 stimulated NF-B activity
and IL-6 gene transcription in FSDC and DC2.4. We were able to rule out
an essential role for TRAF3/5 heterodimers and Jak3 signaling by
showing that a hCD40 protein lacking the most COOH-terminal 15 amino
acids that are critical for binding these proteins (15, 16, 18, 39) had
the ability to induce IB- and IL-6 promoter activities. By
contrast a hCD40 protein carrying a single amino acid substitution T254A (hCD40T254A) that abolishes binding of TRAF2 in addition to
TRAF3/5 and Jak3 (16, 39) was unable to induce IB- promoter activity. Furthermore, this mutant displayed a markedly reduced stimulation of IL-6 promoter activity in response to anti-CD40 (1.5-fold, compared with a 5-fold response with wild type hCD40). Our
data are therefore in agreement with the report by Lee et al. (16), who showed that binding of TRAF3/5 and Jak3 was
dispensable for CD40-induced activation of NF-B, JNK, and ICAM-1
promoter activity. However, other investigators have suggested that
TRAF2 is dispensable for NF-B activation (15), moreover at least two
independent groups have reported that hCD40 carrying the T254A mutation
can support activation of NF-B (18, 44). These latter studies
suggested a role for TRAF6 as a potent activator of CD40-induced NF-B activation since binding of TRAF6 does not require T254. The
residual 1.5-fold induction of IB- promoter activity that we
observed in anti-CD40 stimulated hCD40T254A transfected FSDC may be due
to TRAF6 signaling. Tsukamoto and colleagues (17) recently showed that
TRAF2 and TRAF6 link CD40 to NF-B via distinct signaling pathways.
TRAF2 utilizes a pathway that requires activation of the
NF-B-inducing kinase (NIK), by contrast TRAF6 is able to activate
NF-B independently of NIK. Hence it is possible that the observed
differences in the apparent requirement for TRAF2 or TRAF6 by different
investigators could lie in the differential usage of the
NIK-dependent and -independent signaling pathways. Our
data, which include demonstration that dominant negative TRAF2 inhibits
CD40 stimulation of NF-B activation and IL-6 gene transcription while dominant negative TRAF6 has only a minor effect on these responses, indicate that the TRAF2/NIK pathway may predominate for CD40
activation of NF-B and IL-6 gene transcription in DCs.
Analysis of NF-B DNA binding events induced by anti-CD40 provided a
clue as to why induction of IL-6 was transient and was proceeded by a
loss of basal expression. The NF-B site of the IL-6 promoter
overlaps with a binding site for CBF1 which can repress
NF-B-dependent transcription driven by p50:p65
heterodimers (37, 38). We found that anti-CD40 treatment of FSDC
generated a prolonged elevation of CBF1 DNA binding while induction of
p50:p65 binding was transient. Since overexpression of CBF1 was able to repress basal and anti-CD40 induced IL-6 promoter activity, we suggest
that the prolonged stimulation of CBF1 activity and the short-lived
activation of NF-B and
AP-12 eventually results in a
repression of IL-6 gene transcription. However, the rapid loss of IL-6
transcript between 24 and 48 h post-stimulation of cells also
implies that IL-6 mRNA must be rapidly degraded in FSDC. The short
half-life of the transcript coupled with repression of IL-6 promoter
function by CBF1 is therefore the most likely explanation for the loss
of IL-6 transcript in FSDC stimulated via CD40 for longer than 24 h. Transient induction of IL-6 in CD40 stimulated DCs may be of great
physiological importance. DCs exposed to IL-6 acidify their endosomes,
which results in altered antigen processing leading to priming of T
cell responses against cryptic determinants (10). Prolonged autocrine
stimulation of DCs by IL-6 could therefore result in the generation of
anti-self immunity and propagation of autoimmune disease. Autocrine
IL-6 also inhibits the tolerogenic function of CD8+ DC by
down-regulating IFN-R expression and thereby reducing the ability of
DC to metabolize tryptophan and initiate T cell apoptosis (11). It is
therefore tempting to speculate that the transient induction of IL-6
expression by CD40 stimulated DCs enables activation of naive T cells
but also protects against the generation of anti-self immunity. Further
understanding of the regulatory events that control IL-6 gene
transcription in DCs may lead to the development of experimental
strategies for attenuating DC function in pathological conditions and
in the production of vaccines.
| |
ACKNOWLEDGEMENTS |
We thank David Brenner, Oliver Eickelberg,
Martin Glennie, Ron Hay, Diane Hayward, Luke O'Neill, and Lawrence
Young for their kind gifts of reporter vectors and expression
constructs. We also thank Steve Lyons and Kenneth Rock for their help
with obtaining the DC2.4 cell line.
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ABSTRACT
INTRODUCTION
