| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Receptor Ligation1
*
Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, PA 19140;
Schering-Plough Laboratory of Immunological Research, Dardilly, France; and
Department of Microbiology and Immunology, Weill Medical College, Cornell University, New York, NY 10021
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
R on macrophages results in suppression of IL-12
production. We show that FcR ligation selectively down-regulates
IL-12 p40 and p35 gene expression at the level of transcription. The
region responsive to this inhibition maps to the Ets site of the p40
promoter. PU.1, IFN consensus sequence binding protein, and
c-Rel form a complex on this element upon macrophage
activation. Receptor ligation abolishes the binding of this
PU.1-containing activation complex, and abrogates p40 transcription. A
dominant-negative construct of PU.1 diminishes IL-12 p40 promoter
activity and endogenous IL-12 p40 protein secretion. Thus, the
specificity of IL-12 down-regulation following receptor ligation lies
in the inhibition of binding of a PU.1-containing complex to the Ets
site of the IL-12 promoter. These findings provide evidence
demonstrating for the first time the importance of PU.1 in the
transcriptional regulation of IL-12 gene
expression. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
from T and NK cells. A number of studies have shown that
animals deficient in IL-12 are more susceptible to infections by
intracellular organisms (1, 2), and conversely that the
addition of exogenous IL-12 can promote protective immune responses to
these organisms (3, 4). Despite its conspicuous role in
driving cellular immune responses, the overproduction of IL-12 can be
detrimental to the host. This is especially evident in cases of acute
endotoxemia (5) or during autoimmune diseases (6, 7), where uncontrolled IL-12 production can exacerbate disease.
Thus, the tight regulation of IL-12 is necessary to allow type I immune
responses to occur without the consequent autoimmune sequelae that can
accompany the overproduction of IL-12.
We (8) and others (9) have demonstrated that
ligating phagocytic receptors on macrophages prevents these cells from
making IL-12 in response to a variety of proinflammatory stimuli. The
down-regulation of IL-12 production was a property that was shared by
all of the phagocytic receptors tested, including the FcRs,
complement receptors, and scavenger receptors. Similarly, the ligation
of CD46 (10), CD47 (11), the histamine
H2 receptors (12), or the
2 adrenergic receptors (13) also
resulted in IL-12 down-regulation. Thus, a down-regulation of IL-12
production may accompany the ligation of a variety of receptors on
macrophages.
The regulation of IL-12 transcription is complex and incompletely
understood. The genes for IL-12 p40 and p35 are independently
regulated. The IL-12 p40 gene is transcriptionally regulated and
expressed only in cells making biologically active IL-12
(14). The transcription of IL-12 p40 has been investigated
by several groups (15, 16, 17, 18, 19, 20). Three potential
transcriptional regulatory elements have been identified in the
proximal regions of the IL-12 p40 promoter. An NF-
B half-site has
been identified at -132/-122 bp of the murine IL-12 p40 promoter
(15). NF-B complexes composed of either p50/p65
or p50/c-Rel bind to this site following cellular activation
to produce IL-12. An Ets site is located at -212/-207 in the human
IL-12 p40 promoter, and this site has been identified as a major
response region to IFN- and LPS stimulation (18, 19). A
third potential site for CCAAT/enhancer binding protein is located
at -80/-72 of the murine p40 promoter (17). In a variety
of experimental systems, both the NF-B site and the Ets site are
required for optimal IL-12 p40 transcription. Deleting or mutating
either of these sites causes a dramatic decrease in IL-12
transcription, suggesting that both of these domains are needed to
drive the regulated transcription of p40.
Our understanding of the regulation of the IL-12 p35 gene has lagged
behind that of p40. In leukocytes, p35 is made in very low levels
unless the cells are primed with IFN-, which results in an increase
in p35 transcription (21). The human p35 promoter contains
several putative transcription factor-binding elements including PU.1,
CCAAT/enhancer binding protein, and GAS (IFN- activation sequence).
The p35 gene appears to initiate transcription from multiple sites
(21, 22), and there is evidence to suggest that the p35
gene may also be regulated at the level of post-translational
processing (23, 24).
PU.1 belongs to the Ets family of DNA binding proteins (25, 26). It is expressed predominantly in macrophages, B cells, and erythroid cells (27, 28). PU.1 plays important but varied roles in the development of hematopoietic cells. A deletion of the PU.1 gene leads to a failure to produce mature B lymphocytes and macrophages (29). However, the over-expression of PU.1 prevents terminal differentiation of hematopoietic cells and leads to the overproduction of erythroblasts (30). It was shown recently, using retroviral transduction of PU.1 cDNA into hematopoietic progenitors derived from PU.1-deficient mice, that a graded expression of PU.1 can lead to distinct cell fates (i.e., a low concentration of PU.1 protein induces the B cell fate, whereas a high concentration promotes macrophage differentiation and blocks B cell development) (31). In many instances the transcriptional activation by PU.1 depends on the cooperation of PU.1 with other transcription factors. These factors can either become physically associated with PU.1 or they can bind to DNA adjacent to the Ets site upon which PU.1 resides (32, 33, 34, 35, 36).
In the present work, we examine the molecular mechanism of IL-12
down-regulation following FcR ligation. We show that the decrease in
IL-12 production is due to a decrease in the transcription of both the
IL-12 p40 and p35 genes. This decrease in transcription maps to the Ets
site found in the p40 promoter. In the present work, we characterize a
transcription complex, consisting of PU.1, c-Rel, and IFN
consensus sequence binding protein
(ICSBP),4 that is
formed on this site following cellular activation. We demonstrate that
the ligation of phagocytic receptors on macrophages results in an
inability to form this complex, and a dramatic decrease in IL-12 p40
transcription.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Bone marrow-derived macrophages (BMM
) were established as
previously described (8). One day before use, fully
differentiated BMM were removed from the original plates, washed,
and allowed to readhere to plastic wells in DMEM containing 10%
heat-inactivated FCS, 2 mM L-glutamine (Mediatech,
Herndon, VA) and 100 U/ml penicillin and 100 µg/ml streptomycin
(Mediatech). The murine macrophage-like cell line RAW264.7 was obtained
from American Type Culture Collection (Manassas, VA), and maintained in
RPMI 1640 (Mediatech) containing 10% FCS, 2 mM L-glutamine
and penicillin/streptomycin. All of the Abs used in the Western blots,
immunoprecipitations, and supershift experiments were purchased from
Santa Cruz Biotechnology (Santa Cruz, CA). Recombinant murine IFN-
was purchased from Genzyme (Cambridge, MA). LPS from Escherichia
coli 0127:B8 was purchased from Sigma (St. Louis, MO).
IgG-opsonized erythrocytes (EIgG) were generated by incubating sheep
erythrocytes (Lampire, Pipersville, PA) with rabbit anti-sheep
erythrocyte IgG (Cappel, Durham, NC) at nonagglutinating titers for 40
min at room temperature, as previously described (37).
EIgG were washed with HBSS (Life Technologies, Grand Island, NY) and
resuspended at 2 x 108 cells/ml and used
the same day.
Macrophage stimulation and receptor ligation
Monolayers of BMM were washed and primed with 100 U/ml of
IFN- for 12 h before stimulation with LPS at a final
concentration of 100 ng/ml, in the presence or absence of opsonized
erythrocytes. EIgG were added immediately before LPS stimulation at a
ratio of 20 erythrocytes per macrophage, which generally resulted in a
binding of 1–3 erythrocytes per macrophage after 1 h. RAW264.7
cells were primed with IFN- at a final concentration of 200 U/ml and
stimulated with LPS at a final concentration of 1 µg/ml. Erythrocytes
were added at the same ratio as for BMM.
Plasmids
A 3300-bp and a 220-bp fragment of the human IL-12 p40 promoter
were cloned into the luciferase reporter construct pXP2
(16). A genomic fragment of the TNF-
gene derived from
nucleotides 2849 through 4142 of clone M16441 was cloned into the
SmaI site of the pXP2 luciferase reporter plasmid by blunt
ended ligation. A 1143-bp genomic fragment of the human IL-12 p35
promoter derived from the clone AF050083 corresponding to nucleotide
positions 532 through 1675 was cloned into the pXP2 luciferase vector
as a BamHI-PstI fragment. The NF-B luciferase
plasmid was purchased from Stratagene (La Jolla, CA; catalog number
219078). The PU.1 dominant-negative mutant plasmid was kindly provided
by Michael L. Atchison (University of Pennsylvania, School of
Veterinary Medicine, Philadelphia, PA) (38). All plasmids
were isolated using the Qiagen Endotoxic free kit (Qiagen,
Valencia, CA).
Transfections
RAW264.7 cells stably transfected with a plasmid containing the
luciferase gene driven by a 3.3-kbp fragment of the IL-12 p40 promoter
were generated as previously described (16). Transient
transfections were performed by electroporation as previously described
(16). Briefly, for each condition 0.4 ml of a cell
suspension containing 1 x 107 cells was
mixed with 50 µg total DNA and electroporated in 0.45-cm
electroporation cuvettes (Gene Pulser II; Bio-Rad, Hercules, CA) at 960
microfarads and 340 V in RPMI 1640 without serum. Transfected cells
from the different cuvettes were combined and resuspended in RPMI 1640
containing 10% FCS and 10 µM chloroquine. Cells were added to
24-well plates and incubated for 48 h before harvesting. To
measure luciferase activity, cells were pelleted by centrifugation and
resuspended in 100 µl lysis buffer (20 mM tricine, 1.07 mM
MgCO3, 2.67 mM MgSO4, 0.1
mM EDTA, 33.3 mM DTT, 270 µM coenzyme A, 530 µM ATP, 470 µM
D-luciferine potassium salt). For cotransfection experiments with the
PU.1 dominant-negative mutant, transfected cells from each cuvette were
plated separately and luciferase activity was normalized by
cotransfection of 3 µg pCMV--galactosidase plasmid. Lysates were
used for both luciferase and -galactosidase assays
(16).
Cytokine secretion was measured by ELISA, using appropriately diluted culture supernatants. IL-12 p40 was measured using mAbs C15.6 (anti-murine IL-12 p40) and C17.8 (biotinylated anti-murine IL-12 p40) as ELISA capture and detection Abs, respectively, according to protocols provided by PharMingen (San Diego, CA). Recombinant murine IL-12 (Genzyme) was used as a standard.
RNase protection
RNase protection was performed using the mCK-2 RiboQuant
Multiprobe RNase Protection Assay system from PharMingen according to
the manufacturer’s instructions. A total of 10 µg RNA was used for
each determination. The intensity of all bands was determined by
densitometry after normalizing to the L32 gene. Gels from three
distinct experiments were analyzed and the mean fold decrease following
FcR ligation was determined.
Nuclear extraction
Nuclear extractions for Western analysis and for EMSA assays were performed according the method of Schreiber et al. (39). Briefly, 5–10 x 106 cells were washed and resuspended in 600 µl of buffer containing 10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 1 mM DTT, and 0.5 mM PMSF for 15 min on ice. Cells were lysed in 0.6% Nonidet P-40 with vortexing for 10 s. The homogenate was centrifuged for 30 s in a microfuge and the nuclear pellet was resuspended in ice-cold buffer containing 20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF at 4°C for 15 min with rocking. Following centrifugation in a microfuge for 5 min, the supernatant was either used immediately or frozen at -70°C.
Western blotting and immunoprecipitation
SDS-PAGE was performed according to Laemmli (40)
with 20–30 µg of nuclear extract. Western analysis was performed as
described (18). Gels were electroblotted to PVDF membranes
(Millipore, Bedfore, MA) and blocked in 5% milk in Tris buffer, pH
8.0. Primary Abs were added at the concentration of 1 µg/ml in Tris
buffer containing 1% milk powder for 1 h at room temperature.
After extensive washing, secondary Ab conjugated to HRP was added at a
1:5000 dilution in 1% milk. After extensive washing, blots were
subjected to ECL detection (Amersham Pharmacia Biotech, Piscataway,
NJ). For immunoprecipitation analysis nuclear extracts (100 µg)
prepared from unstimulated or IFN-/LPS-stimulated cells were
precleared for 1 h at 4°C using 15 µl of protein A-agarose
(Santa Cruz Biotechnology). Extracts were immunoprecipitated with 2
µg/ml of anti-PU.1 mAb (Santa Cruz Biotechnology) plus 15 µl
protein A-agarose for 3 h at 4°C. Precipitated protein was
washed five times in buffer (10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM
EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF, 10 mM NaF, 5 mM
Na3VO4, 10 µg/ml
leupeptin, 10 µg/ml pepstatin A). The final pellet was resuspended in
60 µl of SDS sample buffer containing 5% 2-ME, boiled for 5 min, and
electrophoresed on 12% SDS-PAGE.
EMSA
EMSA and supershifts were performed as described previously (18). The sequence spanning the -222/-196 region of the human p40 promoter 5'-CAAAAGTCATTTCCTCTTAGTTCAT-3', was used as a probe for PU.1 binding.
Nuclear run-on assays
The isolation of nuclei and in vitro transcription in the
presence of [32P]UTP (3000 Ci/mmol) were
performed as previously described (18).
[32P] pulse-labeled nuclear RNA was hybridized
for 2 days at 60°C to prehybridized nylon filters to which 500 ng of
denatured cDNA corresponding to the coding regions of IL-12 p35, IL-12
p40, and TNF- had been immobilized using a slot blot apparatus
(Hoeffer Scientific, San Francisco, CA). After hybridization, filters
were washed twice at room temperature with 2x SSC containing 0.1% SDS
for 15 min at 50°C, and once with 0.1x SSC containing 0.1% SDS for
30 min at 50°C. Hybridization was quantified using ImageQuant
software on a PhosphorImager 44581 (Molecular Dynamics, Sunnyvale,
CA).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
R ligation
We have previously shown that the ligation of phagocytic receptors
on macrophages prevented their production of IL-12 in response to
proinflammatory mediators, such as LPS (8). In the present
work, to examine the mechanism of this effect, we used macrophages that
were stimulated with LPS in either the presence or absence of EIgG to
ligate the FcR. Macrophages grown in medium alone without stimuli
produced minimal cytokines, as expected (Fig. 1
). LPS treatment of IFN--primed
macrophages induced the production of a variety of cytokines including
IL-10, IL-1, IL-1, IL-6, migration inhibitory factor (MIF), and
IL-12 p35 and p40. The simultaneous ligation of FcR on parallel
monolayers of stimulated macrophages resulted in marked and specific
reduction in mRNA encoding the two subunits of IL-12, p35, and p40
(Fig. 1). The mRNA levels for many other cytokine genes was either
unchanged or minimally decreased following FcR ligation (Fig. 1).
Densitometric quantitation of three separate RNase protection assays
indicated that whereas IL-12 mRNA levels decreased by >90%, the mRNA
levels for MIF, IL-6, and IL-1RA were not significantly diminished
following FcR ligation. A single other gene, IL-10, was increased
following FcR ligation, as previously reported (37).
The addition of control unopsonized erythrocytes, which do not bind to
specific macrophage receptors, did not affect cytokine mRNA production.
A similar alteration in cytokine production was observed when soluble
immune complexes (OVA/OVA) were used to ligate FcR (data not
shown).
|
/LPS treatment (Fig. 2).
Macrophages stimulated in the presence of EIgG exhibited a decrease in
the transcription of both the IL-12 p40 and p35 genes (Fig. 2).
Transcription of IL-12 in the presence of unopsonized erythrocytes was
the same as that observed in macrophages treated with IFN-/LPS
alone. As a control for these studies, the transcription of TNF- was
also analyzed and found to be minimally reduced following FcR
ligation (Fig. 2). Thus the ligation of FcR on stimulated
macrophages specifically prevented the accumulation of mRNAs for IL-12
p40 and p35, and this regulation occurred at the level of gene
transcription.
|
R ligation
To further examine the regulation of IL-12 transcription following
FcR ligation, an extended (3.3 kbp) segment of the human IL-12 p40
promoter was cloned immediately upstream of a luciferase reporter gene
and the construct was stably transfected into the RAW264.7
macrophage-like cell line, as previously reported (16). To
show that RAW264 cells were appropriate recipient cells for these
assays, endogenous IL-12 from these cells was measured by ELISA
following their stimulation with IFN-/LPS. Stimulated cells made
relatively high levels of IL-12 p40 and these levels were dramatically
reduced when cells were stimulated in the presence of FcR ligation
(Fig. 3A). Thus, like primary
macrophages, these cells respond to LPS stimulation by secreting IL-12,
and they also react to receptor ligation by decreasing IL-12 synthesis,
indicating that they are appropriate cells for use in these assays.
Luciferase activity of the stably transfected cells was measured
following stimulation with LPS in the presence or absence of FcR
ligation. Stimulated cells made relatively high levels of luciferase
and this activity was markedly diminished following receptor ligation
(Fig. 3B). Control unopsonized erythrocytes failed to
abrogate luciferase induction. Thus the RAW264.7 cells used in these
assays largely recapitulate the responses of primary cells to LPS
stimulation, and luciferase activity from these stably transfected
cells is an accurate reflection of IL-12 transcription.
|
and then
stimulated with LPS. Stimulation of primed cells with LPS resulted in
substantial luciferase activity driven by the extended (3.3 kbp) IL-12
p40 promoter. This activity was significantly diminished by ligating
the FcR with EIgG (Fig. 4A), although the effect was
not as dramatic as that observed with stably transfected cells. Control
unopsonized erythrocytes had no effect on LPS-driven luciferase
activity. LPS treatment also stimulated the production of luciferase
driven by the p35 promoter, and this activity was also diminished by
FcR ligation (Fig. 4B), although not as much as the p40
promoter. To identify the promoter regions involved in IL-12
down-regulation, the IL-12 p40 promoter was truncated from 3.3 kbp to
222 bp 5' of the transcription initiation site. This truncated
promoter, which includes three of the putative transcription factor
binding sites of the p40 promoter is the minimal promoter capable of
driving IL-12 transcription in response to IFN- and LPS
(16). This minimal promoter was sufficient to drive
luciferase production, albeit not as well as the extended promoter
(Fig. 4C). However, the ligation of phagocytic receptors
down-regulated luciferase activity to an extent that was comparable to
that observed with the extended promoter. These results suggest that
the down-regulation of IL-12 transcription following receptor ligation
is mediated by the proximal 222 bases of the IL-12 p40 promoter. As
controls for these studies, luciferase driven by the TNF- promoter
and luciferase driven by a consensus NF-B sequence were examined in
parallel. Neither TNF- nor NF-B-driven luciferase activity was
significantly diminished by FcR ligation (Fig. 4, D and
E, respectively). These results demonstrate that the
ligation of phagocytic receptors causes a selective decrease in the
transcription of the IL-12 p40 and p35 genes.
|
R ligation
To further examine the decrease in IL-12 transcription following
FcR ligation, Western blotting analysis was performed to measure the
nuclear translocation of a variety of transcription factors that have
been reported to interact with the IL-12 p40 promoter. Nuclear extracts
were isolated 4 h following cellular activation in the presence or
absence of FcR ligation. The nuclei of unstimulated cells contained
abundant ets-2 but only modest amounts of PU.1 and ICSBP. Little or no
detectable levels of IRF-1 or NF-B transcription factors were
detected in the nuclei of unstimulated cells (Fig. 5, lane 1). When cells were
stimulated with IFN-/LPS all seven of the factors examined were
detected in high levels in nuclear extracts (Fig. 5, lane
2). The ligation of FcR on stimulated cells resulted in a
decrease in the levels of PU.1, ICSBP, and IRF-1 (Fig. 5, lane
3). This decrease was specific to these factors, as the levels of
the three NF-B members (c-Rel, p50, p65) were not
diminished by FcR ligation, and were detected in levels that were
comparable to those observed in cells stimulated with IFN-/LPS alone
(Fig. 5, lane 2 vs lane 3). Consistent with the
nuclear accumulation of NF-B in activated cells, the cytoplasmic
extracts of unstimulated cells contained ample IB, which was rapidly
degraded following IFN-/LPS stimulation, as previously reported
(41). The degradation of cytoplasmic IB was not
reversed by FcR ligation. Thus FcR ligation does not influence
the extent nor the kinetics of NF-B nuclear translocation. However,
FcR ligation did exert a dramatic effect on the nuclear levels of
PU.1, ICSBP, and IRF-1, which were severely reduced following FcR
ligation.
|
R ligation
EMSA were performed using a 27-base probe corresponding to
nucleotides -222/-196 surrounding the Ets domain (-212/-207) of the
IL-12 p40 promoter. In resting cells a single factor bound to this site
(Fig. 6, lane 2). This factor
has previously been identified to be PU.1 (18). In cells
stimulated with IFN-/LPS, a second higher-order complex was formed
on this site (Fig. 6, lane 3, arrow designated
PU.1+). The ligation of FcR resulted in a
dramatic loss of transcription factor binding to this probe (Fig. 6A, lane 4). Neither the lower (resting) band nor
the higher (activation complex) band was detected in these extracts. As
a control, extracts from IFN-/LPS stimulated cells incubated with
unopsonized erythrocytes were analyzed and found to behave similarly to
LPS-stimulated cells, showing both the resting band and the activation
complex (Fig. 6A, lane 5). Supershift assays were
performed to characterize the complex formed on the Ets-2 site
following cellular activation. Ab to PU.1 caused a dramatic decrease in
the binding of all factors to this site, indicating that both the lower
and the upper activation complex are composed of PU.1, and furthermore
that interfering with PU.1 abolishes all DNA binding activity to this
site. The addition of Abs to ICSBP or c-Rel increased the
intensity of the lower band relative to the intensity of the upper band
(Fig. 6A). Abs to several of the other transcription factors
tested did not affect the intensity of either band, indicating that
this increase was specific to ICSBP and c-Rel. These results
suggested that the activation complex formed on this site contains
ICSBP, and c-Rel in addition to PU.1. This conclusion was
supported by immunoprecipitation experiments, where Ab to PU.1 was used
to immunoprecipitate the PU.1 complex from the nuclei of stimulated
cells. This complex was subsequently analyzed by Western blot under
denaturing and reducing conditions. As shown in Fig. 6B, Ab
to PU.1 precipitated not only PU.1, but also ICSBP and
c-Rel. To determine the step at which PU.1 was
down-regulated, we examined the steady-state mRNA expression of PU.1
following FcR ligation. PU.1 mRNA was constitutively expressed and
was not affected at all by FcR cross-linking, whereas both IL-12 p40
and p35 mRNA expression were characteristically inhibited (Fig. 6C), suggesting that FcR ligation targets PU.1 subsequent
to mRNA transcription.
|
). Mutations
encompassing the Ets-2 site abrogated the binding of both complexes,
whereas mutations outside of this site affected neither complex (Fig. 7). Importantly, all mutations that prevented the binding of PU.1
(lower complex) also prevented the formation of the upper
PU.1+ complex, suggesting that the formation of
the activation complex depends on PU.1 binding to the DNA. Furthermore,
there were no mutations that affected only the binding of the
activation complex, indicating that flanking sequences adjacent to the
Ets element were not required for the binding of the activation
complex.
|
To further correlate the decrease in IL-12 production with
alterations in PU.1 activity, a dominant-negative construct of PU.1
(PU.1-DN) was cotransfected into RAW264.7 cells along with a luciferase
construct driven by the IL-12 p40 promoter. Transfected cells were
stimulated with IFN-/LPS to induce the production of luciferase
driven by the IL-12 p40 promoter. In cells cotransfected with the
PU.1-DN construct, IL-12 p40 promoter-driven luciferase activity was
decreased to near background levels (Fig. 8A). Control cells transfected
with the vector alone (pCB6) were fully capable of expressing
IL-12-driven luciferase activity. The inhibition of transcription by
PU.1-DN was specific to IL-12 p40, because neither the TNF-
promoter-driven luciferase activity (Fig. 8B) nor NF-B
(Fig. 8C) was diminished by this construct. Furthermore, the
sequence element(s) responsive to the inhibitor appears to be located
within the proximal 222 bp of the IL-12 p40 promoter, because similar
to the 3.3-kb parental construct, the -222 construct was also
inhibited by ectopic expression of the PU.1-DN (Fig. 8D).
Thus IL-12 p40 transcription depends on PU.1 binding to the Ets site of
the IL-12 p40 promoter, and the inhibition of this binding prevents
IL-12 p40 transcription.
|
It is generally believed that in transient transfections, the
reporter gene is not usually associated with a particular chromatin
structure like the endogenous gene. To determine whether the observed
effects of the PU.1-DN on the IL-12 p40 reporter gene in RAW264.7 cells
could also be exerted on the endogenous IL-12 p40 gene, we transfected
RAW cells with the p40 reporter gene with or without the PU.1-DN.
Cell-free supernatants were collected following appropriate
stimulation, and the endogenously produced murine IL-12 p40 and TNF-
were measured by ELISA. As shown in Fig. 9, the synthesis of endogenous mIL-12 p40
protein was inhibited by 
70% by PU.1-DN, whereas murine TNF- was
not affected. Thus, interfering with PU.1 activity leads to a selective
inhibition of both endogenous and exogenous IL-12 p40 gene
expression.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
R ligation as a model of phagocytic receptor interaction. We show
that the decrease in cytokine production following receptor ligation is
specific to IL-12 p40 and p35, occurs at the level of gene
transcription, and involves a lack of binding of a large transcription
complex to the Ets element in the IL-12 p40 promoter. These
observations begin to explain at the molecular level the
down-regulation of IL-12 production following receptor-mediated
phagocytosis.
We have taken several experimental approaches to show that the
receptor-mediated down-regulation of IL-12 occurs at the level of
transcription. These approaches included RNase protection, nuclear
run-on, and luciferase reporter assays. In all three of these
experimental systems, the ligation of FcRs had a profound inhibitory
effect on IL-12 p40 gene transcription, often reducing transcription to
background levels. Because the IL-12 p40 gene is expressed only in
cells making biologically active IL-12 and because its transcription
has been most closely correlated with IL-12 p70 production, the
regulation of p40 was the initial focus of this work. We show that
IL-12 p40 production was inhibited following FcR ligation. However,
we also analyzed the transcription of IL-12 p35 following receptor
ligation. Although this gene is expressed in much lower levels than
p40, its expression was also significantly decreased by FcR
ligation. By the luciferase assay, p35 promoter activity was decreased
by 50%, and by both RNase protection and nuclear run-on assays the
levels were also substantially reduced. Thus the ligation of FcR on
macrophages decreases the transcription of both the IL-12 p40 and IL-12
p35 genes. The decrease in transcription was specific to IL-12. By
RNase protection assays several other genes, including IL-1,
IL-1, IL-6, and MIF, continued to be transcribed following FcR
ligation (Fig. 1
). Similarly the transcription of TNF-, measured by
either nuclear run-on or luciferase expression was minimally affected
by FcR ligation. Thus receptor ligation does not render macrophages
globally refractory to proinflammatory stimuli; rather it specifically
inhibits IL-12 production.
Nuclear extracts were analyzed 4 h after receptor ligation by
Western blotting to determine whether the accumulation of transcription
factors was affected by FcR ligation. The levels of two IRFs, ICSBP
and IRF-1, were markedly diminished in the nuclei of cells following
FcR ligation. This observation is consistent with previous studies
showing that knockout mice lacking either of these factors were
defective in their ability to produce IL-12 p40 (2).
Similarly, our recent studies (42) demonstrate that the
overexpression of ICSBP could increase IL-12 p40 transcription. Thus
the IRFs are a vital component of IL-12 p40 transcription, and FcR
ligation can functionally mimic the genetic deficiencies in the IRFs. A
third transcription factor was also dramatically affected by FcR
ligation. This was PU.1, a member of the Ets family of transcription
factors. An Ets site is located at -212 of the human IL-12 p40
promoter. This site with flanking sequences (-292 to -196) has
previously been shown to be an important element in controlling the
transcription of IL-12 in the RAW264.7 macrophage cell line
(17). The extended Ets element binds to a high m.w.,
multicomponent complex named F1 (18). In RAW cells, the F1
complex appears to contain a number of transcription factors including
Ets-2, IRF-1, c-Rel, and ICSBP. The over expression of some
of these factors is sufficient to activate the IL-12 p40 promoter
through the Ets site independently of the signals provided by LPS and
IFN- (19, 42). However, in primary macrophages an F1
complex has not been identified. In the present study we describe a
smaller complex induced by IFN-/LPS that binds to the Ets element
but requires less physical space (-222 to -196). In addition to PU.1
this complex also contains ICSBP and c-Rel. We were unable
to detect IRF-1 in this complex by either EMSA or Western analysis. The
nature of the differences between the transformed macrophage cell line
and primary macrophages used in the present work is presently
unclear.
We have taken three different experimental approaches to examine the
binding of transcription factors to the Ets element of the IL-12
promoter, and to determine their role in controlling IL-12 production
following receptor ligation. These approaches included the following:
1) nuclear translocation of putative Ets-binding transcription factors,
2) EMSA using the Ets element of the p40 promoter, and 3) a
dominant-negative construct encoding the DNA binding domain of PU.1,
which lacks the transactivation domain of the wild-type molecule. All
of these approaches pointed to the critical role of PU.1 in activating
IL-12 gene transcription. In the p40 promoter, PU.1 occupies the Ets
site even in resting cells. Resting cells do not transcribe p40 and,
therefore, in this state PU.1 is not a transcriptional activator.
However, in IFN-/LPS-activated cells a larger-molecular-weight
complex is formed on this site (Fig. 6). This activation complex
consists of not only PU.1 but also ICSBP and c-Rel. This was
determined by EMSA coupled with the use of Abs specific to ICSBP and
c-Rel, which revealed a decrease in the intensity of the
upper (activation) complex relative to the lower (PU.1) complex,
indicating that the Abs may have disrupted the higher-order structure
of the activation complex. In addition, an anti-PU.1 Ab could
immunoprecipitate a complex that also contained ICSBP and
c-Rel, further supporting the EMSA data. We hypothesize that
the formation of the PU.1-containing activation complex is necessary
for IL-12 p40 transcription, and that blocking the formation of this
complex by FcR ligation prevents IL-12 biosynthesis. We could
experimentally mimic this blockade by cotransfecting cells with a
dominant-negative construct encoding the DNA binding domain of PU.1.
Cells were transfected with this construct along with a reporter
plasmid driven by the IL-12 p40 promoter. The inhibition of PU.1
binding to the Ets site of the IL-12 p40 promoter by the
dominant-negative construct prevented the synthesis of luciferase,
indicating that the Ets site is a critical element in the regulation of
IL-12.
It should be pointed out that while the formation of the activation
complex on the Ets element was required for IL-12 p40 transcription, it
is probably not sufficient. Previous studies have shown a requirement
for NF-B in driving IL-12 p40 transcription (15), and
recent studies by Sanjabi et al. (43) have shown that mice
lacking c-Rel fail to produce IL-12. We hypothesize that the
Ets site cooperates with the NF-B site to drive IL-12 p40
transcription. This could be achieved through several hypothetical
ways. For example, c-Rel has been shown to be present in
both the ets-related complex and the downstream NF-B complex
(18, 19). It could be a bridging molecule to link the two
complexes. In this case, one would predict a drastic disruption of the
interaction between the two sites if the NF-B site to which
c-Rel binds is mutated. Alternatively, ICSBP could play an
adaptor’s role by virtue of its known interactions with PU.1, which
binds to both the ets site and the PU.1 site immediately adjacent to
the NF-B site (18, 19). In our hands, FcR ligation
did not affect IB degradation or the translocation of NF-B (p65,
p50, and c-Rel) into the nucleus of activated cells (Fig. 5). Furthermore, luciferase driven by a consensus NF-B site was not
decreased by FcR ligation (Fig. 4E). Thus, global
alterations in NF-B do not account for the ablation of IL-12
production following FcR ligation
Our data for the first time demonstrate a reciprocal link between
FcR, an important mediator in both innate and adaptive immunity, and
PU.1, a transcription factor indispensable for myeloid differentiation.
The molecular basis of FcR-mediated selective targeting of PU.1 is
not presently understood, but these studies are on-going. Our data
indicate that mRNA synthesis of PU.1 is not affected by FcR ligation
(Fig. 6C), suggesting that the PU.1 deficiency occurs
downstream at the protein level. The mechanism of PU.1 inactivation may
involve an alteration in its phosphorylation, because the
phosphorylation status of PU.1 has been reported to affect its
association with other transcription factors and its transcriptional
activity (32).
In summary, we have shown that FcR ligation leads to an inhibition
of IL-12 transcription and we conclude that the primary mechanism
responsible for this decreased transcription lies in the disruption of
the formation of a PU.1-containing complex. These results suggest that
an important component of the regulation of IL-12 biosynthesis in
myeloid cells lies with the myeloid-specific transcription factor,
PU.1.
|
Footnotes |
|---|
2 D.M.M. and X.M. are co-senior authors.
3 Address correspondence and reprint requests to Dr. David M. Mosser at his current address: Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742.
4 Abbreviations used in this paper: ICSBP, IFN consensus sequence binding protein; BMM, bone marrow-derived macrophages; EIgG, IgG-opsonized erythrocytes; IRF-1, IFN regulatory factor 1; PU.1-DN, dominant-negative construct of PU.1; PU.1-DNM, PU.1-DN mutant; MIF, migration inhibitory factor.
Received for publication October 18, 2000. Accepted for publication January 17, 2001.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
production and lethality in lipopolysaccharide-induced shock in mice. Eur. J. Immunol. 25:672.[Medline]
2-agonists prevent Th1 development by selective inhibition of interleukin 12. J. Clin. Invest. 100:1513.[Medline]
B half-site. Mol. Cell Biol. 15:5258.[Abstract]
in monocytic cells. J. Exp. Med. 183:147.B and Ets transcription factors in Epstein-Barr virus-transformed B cells and macrophages. J. Biol. Chem. 273:6431.-dependent inducible expression of the human interleukin-12 p35 gene in monocytes initiates from a TATA-containing promoter distinct from the CpG-rich promoter active in Epstein-Barr virus-transformed lymphoblastoid cells. Blood 91:4645. 3' enhancer activity. Mol. Cell Biol. 12:368. receptor type I. J. Exp. Med. 188:217.B by phosphorylation of its inhibitor IB. Nature 344:678.[Medline]
-inducible transcription factor, IFN consensus sequence binding protein (ICSBP), stimulates IL-12 p40 expression in macrophages. J. Immunol. 165:271.This article has been cited by other articles:
|
|
 |
|
  S. Divanovic, A. Trompette, L. K. Petiniot, J. L. Allen, L. M. Flick, Y. Belkaid, R. Madan, J. J. Haky, and C. L. Karp Regulation of TLR4 signaling and the host interface with pathogens and danger: the role of RP105 J. Leukoc. Biol., August 1, 2007; 82(2): 265 - 271. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. K. Polumuri, V. Y. Toshchakov, and S. N. Vogel Role of Phosphatidylinositol-3 Kinase in Transcriptional Regulation of TLR-Induced IL-12 and IL-10 by Fc{gamma} Receptor Ligation in Murine Macrophages J. Immunol., July 1, 2007; 179(1): 236 - 246. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Ma, S. Mishra, K. Gee, J. P. Mishra, D. Nandan, N. E. Reiner, J. B. Angel, and A. Kumar Cyclosporin A and FK506 Inhibit IL-12p40 Production through the Calmodulin/Calmodulin-dependent Protein Kinase-activated Phosphoinositide 3-Kinase in Lipopolysaccharide-stimulated Human Monocytic Cells J. Biol. Chem., May 4, 2007; 282(18): 13351 - 13362. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. W. Moore, A. R. Cruz, C. J. LaVake, A. L. Marzo, C. H. Eggers, J. C. Salazar, and J. D. Radolf Phagocytosis of Borrelia burgdorferi and Treponema pallidum Potentiates Innate Immune Activation and Induces Gamma Interferon Production Infect. Immun., April 1, 2007; 75(4): 2046 - 2062. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. M. Townsend, I. Gonzalez-Gomez, and J. L. Badger fliP influences Citrobacter koseri macrophage uptake, cytokine expression and brain abscess formation in the neonatal rat. J. Med. Microbiol., December 1, 2006; 55(Pt 12): 1631 - 1640. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Cao, X. Zhang, J. P. Edwards, and D. M. Mosser NF-{kappa}B1 (p50) Homodimers Differentially Regulate Pro- and Anti-inflammatory Cytokines in Macrophages J. Biol. Chem., September 8, 2006; 281(36): 26041 - 26050. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. D. Iankov, M. Pandey, M. Harvey, G. E. Griesmann, M. J. Federspiel, and S. J. Russell Immunoglobulin g antibody-mediated enhancement of measles virus infection can bypass the protective antiviral immune response. J. Virol., September 1, 2006; 80(17): 8530 - 8540. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Liu and X. Ma Interferon Regulatory Factor 8 Regulates RANTES Gene Transcription in Cooperation with Interferon Regulatory Factor-1, NF-{kappa}B, and PU.1 J. Biol. Chem., July 14, 2006; 281(28): 19188 - 19195. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Ito, H. Kanzler, O. Duramad, W. Cao, and Y.-J. Liu Specialization, kinetics, and repertoire of type 1 interferon responses by human plasmacytoid predendritic cells Blood, March 15, 2006; 107(6): 2423 - 2431. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Fronhofer, M. R. Lennartz, and D. J. Loegering Role of PKC isoforms in the Fc{gamma}R-mediated inhibition of LPS-stimulated IL-12 secretion by macrophages J. Leukoc. Biol., February 1, 2006; 79(2): 408 - 415. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. N. Waggoner, M. W. Cruise, R. Kassel, and Y. S. Hahn gC1q Receptor Ligation Selectively Down-Regulates Human IL-12 Production through Activation of the Phosphoinositide 3-Kinase Pathway J. Immunol., October 1, 2005; 175(7): 4706 - 4714. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Lucas, X. Zhang, V. Prasanna, and D. M. Mosser ERK Activation Following Macrophage Fc{gamma}R Ligation Leads to Chromatin Modifications at the IL-10 Locus J. Immunol., July 1, 2005; 175(1): 469 - 477. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Laine, J. M. Bourhis, S. Longhi, M. Flacher, L. Cassard, B. Canard, C. Sautes-Fridman, C. Rabourdin-Combe, and H. Valentin Measles virus nucleoprotein induces cell-proliferation arrest and apoptosis through NTAIL-NR and NCORE-Fc{gamma}RIIB1 interactions, respectively J. Gen. Virol., June 1, 2005; 86(6): 1771 - 1784. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Ji, J. Masterson, J. Sun, and L. Soong CD4+CD25+ Regulatory T Cells Restrain Pathogenic Responses during Leishmania amazonensis Infection J. Immunol., June 1, 2005; 174(11): 7147 - 7153. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Qin, J. C. Edberg, A. W. Gibson, G. P. Page, L. Teng, and R. P. Kimberly Differential Gene Expression Modulated by the Cytoplasmic Domain of Fc{gamma}RIa (CD64) {alpha}-Chain J. Immunol., November 15, 2004; 173(10): 6211 - 6219. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. C. Marie, F. Saltel, J.-M. Escola, P. Jurdic, T. F. Wild, and B. Horvat Cell Surface Delivery of the Measles Virus Nucleoprotein: a Viral Strategy To Induce Immunosuppression J. Virol., November 1, 2004; 78(21): 11952 - 11961. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Xiong, H. Li, Y. Chen, J. Zhao, and J. C. Unkeless Interaction of TRAF6 with MAST205 Regulates NF-{kappa}B Activation and MAST205 Stability J. Biol. Chem., October 15, 2004; 279(42): 43675 - 43683. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Mitsuhashi, J. Liu, S. Cao, X. Shi, and X. Ma Regulation of interleukin-12 gene expression and its anti-tumor activities by prostaglandin E2 derived from mammary carcinomas J. Leukoc. Biol., August 1, 2004; 76(2): 322 - 332. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. H. Sulahian, P. A. Pioli, K. Wardwell, and P. M. Guyre Cross-linking of Fc{gamma}R triggers shedding of the hemoglobin-haptoglobin scavenger receptor CD163 J. Leukoc. Biol., July 1, 2004; 76(1): 271 - 277. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Schmidt, J. Bies, T. Tamura, K. Ozato, and L. Wolff The interferon regulatory factor ICSBP/IRF-8 in combination with PU.1 up-regulates expression of tumor suppressor p15Ink4b in murine myeloid cells Blood, June 1, 2004; 103(11): 4142 - 4149. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Zhou, H. Xiong, H. Li, S. E. Plevy, P. D. Walden, M. Sassaroli, G. D. Prestwich, and J. C. Unkeless Microtubule-Associated Serine/Threonine Kinase-205 kDa and Fc{gamma} Receptor Control IL-12 p40 Synthesis and NF-{kappa}B Activation J. Immunol., February 15, 2004; 172(4): 2559 - 2568. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. L. Oleszak, J. R. Chang, H. Friedman, C. D. Katsetos, and C. D. Platsoucas Theiler's Virus Infection: a Model for Multiple Sclerosis Clin. Microbiol. Rev., January 1, 2004; 17(1): 174 - 207. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Romani, C. Montagnoli, S. Bozza, K. Perruccio, A. Spreca, P. Allavena, S. Verbeek, R. A. Calderone, F. Bistoni, and P. Puccetti The exploitation of distinct recognition receptors in dendritic cells determines the full range of host immune relationships with Candida albicans Int. Immunol., January 1, 2004; 16(1): 149 - 161. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Liu, S. Cao, L. M. Herman, and X. Ma Differential Regulation of Interleukin (IL)-12 p35 and p40 Gene Expression and Interferon (IFN)-{gamma}-primed IL-12 Production by IFN Regulatory Factor 1 J. Exp. Med., October 20, 2003; 198(8): 1265 - 1276. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Zhu, K. Rao, H. Xiong, K. Gagnidze, F. Li, C. Horvath, and S. Plevy Activation of the Murine Interleukin-12 p40 Promoter by Functional Interactions between NFAT and ICSBP J. Biol. Chem., October 10, 2003; 278(41): 39372 - 39382. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-D. Ji, I. Tassiulas, K.-H. Park-Min, A. Aydin, I. Mecklenbrauker, A. Tarakhovsky, L. Pricop, J. E. Salmon, and L. B. Ivashkiv Inhibition of Interleukin 10 Signaling after Fc Receptor Ligation and during Rheumatoid Arthritis J. Exp. Med., June 2, 2003; 197(11): 1573 - 1583. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Verbovetski, H. Bychkov, U. Trahtemberg, I. Shapira, M. Hareuveni, O. Ben-Tal, I. Kutikov, O. Gill, and D. Mevorach Opsonization of Apoptotic Cells by Autologous iC3b Facilitates Clearance by Immature Dendritic Cells, Down-regulates DR and CD86, and Up-regulates CC Chemokine Receptor 7 J. Exp. Med., December 16, 2002; 196(12): 1553 - 1561. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Cao, J. Liu, M. Chesi, P. L. Bergsagel, I-C. Ho, R. P. Donnelly, and X. Ma Differential Regulation of IL-12 and IL-10 Gene Expression in Macrophages by the Basic Leucine Zipper Transcription Factor c-Maf Fibrosarcoma J. Immunol., November 15, 2002; 169(10): 5715 - 5725. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Zhao, C. A. Cuff, E. Moss, U. Wille, T. Cyrus, E. A. Klein, D. Pratico, D. J. Rader, C. A. Hunter, E. Pure, et al. Selective Interleukin-12 Synthesis Defect in 12/15-Lipoxygenase-deficient Macrophages Associated with Reduced Atherosclerosis in a Mouse Model of Familial Hypercholesterolemia J. Biol. Chem., September 13, 2002; 277(38): 35350 - 35356. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. A. Butcher and E. Y. Denkers Mechanism of Entry Determines the Ability of Toxoplasma gondii To Inhibit Macrophage Proinflammatory Cytokine Production Infect. Immun., September 1, 2002; 70(9): 5216 - 5224. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Caamano and C. A. Hunter NF-{kappa}B Family of Transcription Factors: Central Regulators of Innate and Adaptive Immune Functions Clin. Microbiol. Rev., July 1, 2002; 15(3): 414 - 429. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. F. Anderson and D. M. Mosser A novel phenotype for an activated macrophage: the type 2 activated macrophage J. Leukoc. Biol., July 1, 2002; 72(1): 101 - 106. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. F. Anderson and D. M. Mosser Cutting Edge: Biasing Immune Responses by Directing Antigen to Macrophage Fc{gamma} Receptors J. Immunol., April 15, 2002; 168(8): 3697 - 3701. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Mason, J. Aliberti, J. C. Caamano, H.-C. Liou, and C. A. Hunter Cutting Edge: Identification of c-Rel-Dependent and -Independent Pathways of IL-12 Production During Infectious and Inflammatory Stimuli J. Immunol., March 15, 2002; 168(6): 2590 - 2594. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Bozza, R. Gaziano, A. Spreca, A. Bacci, C. Montagnoli, P. di Francesco, and L. Romani Dendritic Cells Transport Conidia and Hyphae of Aspergillus fumigatus from the Airways to the Draining Lymph Nodes and Initiate Disparate Th Responses to the Fungus J. Immunol., February 1, 2002; 168(3): 1362 - 1371. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
0.01).