| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814;
Pulmonary Center and Department of Pathology, Boston University School of Medicine, Boston, MA 02118; and
National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
. Like induction of
leishmaniacidal activity, LPS and IFN- synergize to induce ICSBP
mRNA and protein. Deletion analysis of the ICSBP promoter identified
regions that were IFN- responsive, regions that mediate the ability
of LPS and IFN- to activate this promoter synergistically, as well
as regions that normally repress ICSBP transcription. Finally,
exogenous expression of ICSBP, found in previous studies to
down-regulate MHC I gene expression, failed to repress basal or
IFN--induced ICSBP transcription. This demonstrates that ICSBP can
selectively suppress the expression of IFN-responsive genes. These
findings extend in a significant way our understanding of the
regulation of ICSBP by LPS and IFN- and provide important clues as
to its role in macrophage activation. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
) and a trigger signal (e.g., LPS), are required (3). Synergy
between IFN- and LPS in the stimulation of macrophages has been
extended to include the regulation of certain genes (e.g., iNOS) that
contribute to the microbicidal and tumoricidal capabilities of these
cells (reviewed in 7).
The study of genes that are transcriptionally activated by IFN has
identified homologous sequences within their promoters that act as
cis-acting elements to regulate the expression of these
genes. The first sequence of this type was identified by Friedman and
Stark (8) and was designated the IFN consensus sequence
(ICS).4 It was subsequently
established that a sequence within the ICS, termed the
IFN-stimulated response element (ISRE), conferred IFN inducibility on a
heterologous promoters (reviewed in 9). The ISRE can be bound by
members of a growing family of nuclear proteins called IFN regulatory
factors (IRFs) (reviewed in 9). There are currently 10 members in
this family of related proteins, including IRF-1, IRF-2, the
subunit of IFN-stimulated gene factor-3 (ISGF3), and IFN consensus
sequence binding protein (ICSBP). These proteins share significant
homology in their N-terminal domains, which allows for interaction with
a consensus DNA sequence, (G/C)(A)AAAN2–3AAA(G/C)(T/C),
present in the ISRE. There is only limited homology between any of
these proteins at their C-terminal ends.
Despite the fact that they share DNA binding sites, the IRF family
members differ markedly in their abilities to modulate gene
transcription (9). For example, IRF-1 plays a role as a positive
regulator in the expression of genes such as MHC class I and IFN-ß
(10, 11, 12, 13). ISGF3, the DNA binding subunit of the ISGF3 complex, also
acts as a trans-activator (14, 15, 16, 17). In contrast, IRF-2 has
generally been associated with negative regulation of gene expression
(11), although it was recently shown to up-regulate the expression of
certain genes (18). In contrast, ICSBP, whose expression is highly
restricted to cells of monocytic and lymphocytic lineages (19), has
only been reported to down-regulate the expression of a number of
IFN-inducible genes (e.g., MHC class I, 2',5'-oligoadenylate
synthetase, guanylate-binding protein, and ISG-15 genes) (20, 21). The
proper balance of IRF family members may be the most important criteria
in the control of IFN-regulated gene expression. For example, IRF-1 and
IRF-2 compete for the same ISRE, but exert opposing effects on IFN and
MHC class I genes (11, 22).
ICSBP has been shown to form complexes with IRF-1 or IRF-2 both in vivo
and in vitro regardless of the presence of the target DNA. A tighter
ICSBP/IRF-2 complex confers more cooperative DNA binding activity on
both proteins than that of the ICSBP/IRF-1 complex (23). In addition,
the least stable complex, ICSBP/ISGF3, inhibits the DNA-binding
activity of ISGF3 (23). Sharf et al. (24) identified three distinct
functional domains within ICSBP: a DNA binding domain, a
transcriptional repressing domain, and a domain responsible for the
association with other IRFs. In addition to its uniqueness as a
trans-repressor, and its restricted expression in immune
cell types, recent studies using ICSBP knockout mice have revealed the
importance of ICSBP in macrophage activation (25, 26, 27). ICSBP knockout
mice fail to control infection by Toxoplasma gondii and
Leishmania donovani, pathogens that depend on the presence
of highly activated macrophages for their elimination. Moreover, these
studies revealed that defective production of IL-12 p40 and, as a
consequence, limited production of IFN-, underlie their sensitivity
to these pathogens.
Our previous studies demonstrated that IFN-, but not IFN-
/ß,
was a potent inducer of ICSBP mRNA and protein in murine macrophages
(28, 29), and that LPS also induces ICSBP mRNA expression, albeit less
potently than IFN- (30). In this study we sought to analyze the
capacity of LPS and IFN- to regulate ICSBP transcription in
macrophages. Under conditions where we have demonstrated synergistic
induction of leishmaniacidal activity in murine macrophages by IFN-
and LPS (31), the data presented herein demonstrate that ICSBP mRNA and
protein are also induced synergistically. Moreover, analysis of the
ICSBP promoter was conducted to evaluate the effects of LPS and/or
IFN- on ICSBP transcription and the potential role of ICSBP in the
control of its own expression.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
C3H/OuJ and C3H/HeJ mice (female, 5–6 wk old) were obtained from The Jackson Laboratory (Bar Harbor, ME). All mice were used within 1 wk of receipt. Mice were maintained in a laminar flow hood in cages fitted with polyester filter hoods and were fed standard lab food and acid water ad libitum. Macrophages were elicited by i.p. injection of 3 ml of sterile fluid thiogycollate medium (3%) and subsequently harvested by peritoneal lavage 4 days after injection. Cells were cultured at 37°C and 6% CO2 in tissue culture petri dishes (100 mm; Falcon, Lincoln Park, NJ) at about 25 x 106 cells/plate in 15 ml of RPMI 1640 (M. A. Bioproducts, Walkersville, MD) supplemented with 2% FCS, 2 mM glutamine, 30 mM HEPES, 0.4% sodium bicarbonate, and penicillin and streptomycin (100 IU/ml and 100 µg/ml, respectively) as described previously (28, 29). RAW 264.7, a mouse macrophage cell line, was purchased from American Type Culture Collection (Manassas, VA). RAW 264.7 cells were cultured in DMEM (Life Technologies, Gaithersburg, MD) supplemented with 5% FBS (HyClone, Logan, UT), 10 mM HEPES, 10 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C and 5% CO2.
Reagents
Protein-free (<0.008%), phenol/water-extracted
Escherichia coli K235 LPS was prepared by the method of
McIntire et al. (32). Purified, recombinant murine IFN- (1.3 x
107 U/mg) was provided by Genentech (South San Francisco,
CA).
Northern blot analysis
Northern blot analysis was conducted exactly as detailed
previously (28). Blots were probed first with
[-32P]dCTP-labeled ICSBP cDNA probe, and subsequently
with [-32P]dCTP-labeled ß-actin cDNA probe. The
expression of ß-actin mRNA was not modulated by any of the treatments
used. Following hybridization with 32P-labeled probes,
blots were exposed to x-ray film (Kodak X-OMAT AR 5, Eastman Kodak,
Rochester, NY) with Kodak X-Omatic intensifying screens. Blots were
also exposed to storage phosphor screens (Molecular Dynamics,
Sunnyvale, CA) and analyzed using a PhosphorImager (Molecular Dynamics,
model 400A) with ImageQuant software (version 3.0). To correct for
unequal RNA loading onto gels, the ICSBP mRNA signals for each lane
were adjusted relative to its corresponding signal for ß-actin. The
ratio of ICSBP/ß-actin signals was used to calculate fold induction
from control treatments. The size of the labeled RNA species was
determined by comparing migration distances to RNA standards, which
were included in the agarose gel (Life Technologies, Gaithersburg, MD).
Western blot analysis
Nuclear proteins were isolated from macrophages that were lysed under hypotonic conditions with Nonidet P-40 (0.5%) in the presence of protease inhibitors as described previously (29). Western blot analysis for the detection of ICSBP protein was conducted as detailed previously (29). Briefly, equal amounts of protein (5–15 µg) were subjected to SDS-PAGE using the Bio-Rad minigel system (Mini Protean II) and a 12% acrylamide gel. Proteins were transferred electrically to Immobilon-P membranes (Millipore Corp., Bedford, MA). Prestained m.w. markers (Bio-Rad, Richmond, CA) were included in each gel. The membranes were blocked in 5% blocker (Bio-Rad) mixed in Tris-buffered saline for at least 1 h. Detection of ICSBP protein was conducted using the enhanced chemiluminescence (Amersham, Arlington Heights, IL) method according to the manufacturer’s instructions. Hyperimmune anti-ICSBP peptide rabbit serum was diluted 1/800 in blocking buffer and incubated with the membranes for 1 h after blocking. After washing in Tris-buffered saline, the membranes were incubated in a HRP-conjugated secondary Ab (goat anti-rabbit IgG, Bio-Rad), diluted 1/7500 in blocking buffer, for 45 min at 20°C. Membranes were exposed to x-ray film for 0.5–5 min after incubation in HRP substrate.
EMSA
EMSA analysis was performed as we previously described (33).
Nuclear extracts were prepared from approximately 1 x
107 RAW 264.7 cells by the method of Schreiber et al. (34).
All extracts were stored in aliquots at -70°C, and multiple
freeze-thawing cycles were avoided. A portion of the cells was treated
with LPS (5 ng/ml) and/or IFN- (0.25 U/ml) for 3 h before
harvesting. To measure ICSBP DNA-binding activity, a double-stranded
25-bp DNA oligonucleotide (Life Technologies) containing a single copy
of the mouse Ig
-chain 3' enhancer IRF/Ets composite binding site
(5'-GAGGAACTGAAAC-3', the ICSBP binding site is underlined)
was used as both probe and competitor DNA. This composite binding site
was used instead of an ISRE in these experiments because ICSBP binding
to DNA is greatly stabilized in the presence of the transcription
factor PU.1, which binds to the upstream GGAA motif (35). PU.1 is
constitutively expressed in RAW 264.7 cells, and its DNA-binding
activity is not altered by treatment of the cells with either LPS or
IFN- (data not shown).
DNA probes were generated by radiolabeling at the 5' overhangs with
[-32P]deoxynucleotide triphosphates (DuPont-New
England Nuclear, Boston, MA) using E. coli DNA polymerase
Klenow fragment (U.S. Biochemical Corp., Cleveland, OH) as recommended
by the manufacturer. Unincorporated nucleotides were removed using
Sephadex G-25 columns (5 Prime-3 Prime, Boulder, CO). Crude nuclear
extracts (typically 2 µg) were incubated with radiolabeled probe DNA
(0.1 ng, typically 10,000 cpm) in the presence of 2 µg of poly(dI-dC)
(Pharmacia, Piscataway, NJ), 1 mM EDTA, 10 mM Tris-HCl (pH 7.9), 25 mM
glycerol, and 0.5 mM DTT in a final volume of 20 µl at room
temperature for 30 min as we have previously described (36). In
competition experiments, unlabeled DNA was added just before addition
of the extract at a 200-fold molar excess. Binding reactions were
performed at room temperature for 30 min, then a portion of the mixture
(7 µl) was electrophoresed on a 7% nondenaturing, low ionic strength
mobility shift polyacrylamide gel using an electrophoresis buffer
containing 20 mM Tris-HCl (pH 8.0), 20 mM borate, and 0.5 mM EDTA
(0.25x TBE). Gels were dried without fixation and were visualized by
autoradiography.
Plasmids
The pUE5 (the ICSBP expression plasmid), pLK440 (the empty
control vector), pICSBP(351)-CAT and pICSBP(151)-CAT (containing 351
and 151 bp, respectively, of the ICSBP promoter upstream sequences from
the transcriptional starting site), and MHC I-CAT construct,
pLd-1.4K-CAT reporter plasmids were described in detail in
previous studies (20, 37, 38). The C-15 CAT (wild-type) and 5.6 CAT
(IRF site mutant, termed C15 m) reporter constructs containing four
tandem copies of the composite IRF/Ets binding site derived from
the Ig 3' enhancer were provided by Dr. Michael
Atchison (University of Pennsylvania), and were previously
described (39). The control vector pcDNA3.1 was purchased from
Invitrogen (Carlsbad, CA) and was used to maintain a constant amount of
plasmid DNA in each transfection experiment. The deletion plasmids
pICSBP(251)-CAT, pICSBP(236)-CAT, pICSBP(221)-CAT, and pICSBP(179)-CAT,
containing 251, 236, 221, and 179 bp of ICSBP promoter upstream
sequences, respectively, were cloned using PCR methodology. Briefly,
the XbaI-SphI fragment of pICSBP(351)-CAT, which
contains the ICSBP promoter, was replaced with one of four
PCR-amplified fragments. The sense strand primers used to generate the
5' ends of each fragment were: AAT TGC ATG CTT TTG GGG CAG CCC CCT
(251), AAT TGC ATG CCT CCC CCG CCG CCC CCG (236), GGC TGC ATG CCG GAG
TAA AGA GAG AAA (221), and GGC TGC ATG CCA AAA GTG ATT TCT CGG (179). A
common 3' antisense strand primer, ATC CTC TAG ACT GCC CGC GC, was used
to generate all four PCR products. The sequences of all constructs were
confirmed by sequencing analysis (model 373A DNA Sequencing System,
Applied Biosystems, Foster City, CA).
Measurement of reporter gene activity
RAW 264.7 cells were transfected using either Lipofectamine
(Life Technologies) or SuperFect (Qiagen, Valencia, CA). Cells were
first seeded in 60-mm tissue culture plates at 3 x
106 cells/plate and incubated at 37°C in 5%
CO2 for 18–24 h before transfection. For transfections
performed using Lipofectamine, 1 µg of target plasmid was diluted in
300 µl of OPTI-MEM I Reduced Serum Medium (Life Technologies) and
then mixed gently with the mixture of 20 µl of Lipofectamine reagent
and 300 µl of OPTI-MEM I for 30 min at room temperature to allow
DNA-liposome complexes to form. While complexes formed, the cells were
rinsed once with OPTI-MEM I. For each transfection, 2.4 ml of OPTI-MEM
I was added to the Lipofectamine-DNA mixtures, then overlaid onto the
cells and incubated at 37°C in 5% CO2 for 5 h.
Following incubation, 3 ml of growth medium containing 10% FBS was
added without removing the transfection mixture, then incubated
overnight. When cotransfections with both expression and reporter
plasmids were conducted, a total of 5 µg of plasmid was added per
plate. The total DNA amount was kept constant by adding pLK440 plasmid
DNA. The medium was changed, and the cells were treated with the
indicated concentration of medium only, LPS alone, IFN- alone, or
LPS plus IFN- for 8 h. For transfections performed using
SuperFect, a total of 4 µg of plasmid was used per condition. Cells
were transfected according to the manufacturer’s instructions.
Following transfection and treatment with LPS and/or IFN-, cells
were washed with PBS, harvested, and then lysed by freeze-thawing.
Lysates were heated at 65°C for 10 min and were assayed for CAT
activity by either the method of Gorman et al. (40) or by a two-phase
fluor assay as we have previously described (33). The amount of each
lysate used in the CAT assays was equilibrated by protein content
determined using the Bio-Rad protein assay according to the
manufacturer’s instructions.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
synergize to induce ICSBP mRNA and protein in
murine macrophages
As reported previously (28, 30), we found that either IFN- or
LPS can stimulate the expression of ICSBP mRNA in C3H/OuJ macrophages;
two coordinately regulated mRNA transcripts of 1.7 and 3.0 kb were
detected (28, 30). Fig. 1
A
illustrates that IFN- and LPS synergize to induce ICSBP steady state
mRNA in these murine macrophages. As reported previously (30),
treatment of peritoneal exudate macrophages with 1–10 ng/ml LPS has
only a minimal stimulatory effect on the expression of ICSBP mRNA
4 h after treatment. Although a suboptimal concentration of
IFN- (0.25 U/ml) alone induced a significant level of ICSBP mRNA
(
12-fold induction over basal levels), simultaneous stimulation of
macrophages with LPS (1–10 ng/ml) and IFN- (0.25 U/ml) was highly
synergistic. The inset in Fig. 1A shows the results of a
typical Northern blot experiment.
|
53 kDa by Western blot analysis (29). Therefore,
experiments analogous to those described above were conducted to
evaluate the effects of LPS plus IFN- on the induction of ICSBP
protein. Because detection of protein by Western blot analysis relies
on a nonradioactive signal, densitometric technology was employed to
quantify differences in ICSBP protein levels. In untreated macrophages,
ICSBP protein was not detectable (i.e., the failure to detect
measurable signal in Western blots of cells treated with medium alone
precludes the calculation of fold induction). ICSBP expression was
readily detected after 4 h of treatment with 10 ng/ml LPS (Fig. 1
B). In the absence of LPS, as little as 0.25 U/ml IFN-
induced levels of ICSBP protein approximately twice as high as those
induced by 10 ng/ml LPS alone. As was observed for ICSBP mRNA
expression, LPS had a much more dramatic effect on protein levels when
present in combination with IFN-. A steep dose-response relationship
was observed when IFN- and LPS (0.1–1 ng/ml) were present
concurrently. This synergistic effect was maximal at doses of LPS >1
ng/ml.
When present individually, the two stimuli differ in their time course
of ICSBP mRNA induction (Fig. 2). LPS
induces ICSBP mRNA expression with somewhat slower kinetics than
IFN-. In addition, LPS-induced ICSBP mRNA was maintained at high
levels for at least 30 h, while induction by IFN- was more
transient, approaching baseline values approximately 10 h after
stimulation. Synergy between IFN- and LPS was evident only during
the first few hours of treatment, after which mRNA levels declined to
levels similar to those induced by LPS alone and remain elevated at the
level of cells treated with LPS for 30 h poststimulation. To date,
the synergy between IFN- and LPS has been shown using suboptimal
concentrations of IFN-. We also tested for synergistic responses
using 10 U/ml IFN-, an optimal ICSBP-inducing concentration for
murine macrophages (28, 29). Fig. 3
demonstrates that synergy between LPS and IFN- was observed even
when the latter was present at 10 U/ml. To confirm that LPS is
responsible for the observed synergy of ICSBP induction, the effects of
combined LPS and IFN- treatment were compared in macrophages from
the LPS-hyporesponsive (Lpsd) mouse strain, C3H/HeJ,
vs the fully LPS-responsive (Lpsn) C3H/OuJ mouse
strain. As shown in Fig. 4, LPS-induced
effects on ICSBP mRNA induction (either alone or in synergy with
IFN-) were observed only with macrophages obtained from normally
LPS-responsive C3H/OuJ mice and not with those from LPS-hyporesponsive
C3H/HeJ mice.
|
|
|
The effect of combined LPS and IFN- treatment on the
transcriptional capability of the ICSBP promoter was subsequently
evaluated. The macrophage cell line RAW 264.7 was transiently
transfected with a CAT reporter plasmid under the control of a 351-bp
fragment of the ICSBP promoter (ICSBP(351)-CAT), as described
previously (37, 38). In preliminary experiments the RAW 264.7
macrophage cell line was found to respond synergistically to LPS plus
IFN- to produce ICSBP (data not shown). Fig. 5 illustrates that CAT activity was
induced by IFN- (10 U/ml) alone, but not appreciably by LPS alone
(maximum of 50 ng/ml tested). Simultaneous treatment of the transfected
cells with LPS and IFN- resulted in CAT activity significantly
greater than observed with IFN- alone.
|
The transcriptional start (cap) site of the murine ICSBP gene was
identified in a previous study (38). Sequences upstream from the cap
site contained putative TATA and CCAAT box elements at positions -26
and -116 bp, respectively. In addition, additional putative
cis elements were identified: GC boxes (positions -35,
-49, -227), a palindromic IRE5
(referred to as pIRE, position -155), an IRE-like motif (position
-198; see Footnote 5), and an NF-B site (position -531). To
determine the essential cis element(s) that confers maximal
basal or inducible levels of ICSBP promoter activity in murine
macrophages in transient assays, deletion mutants were constructed from
the pICSBP(351)-CAT reporter plasmid that contains the first 351 bp of
the ICSBP promoter upstream sequences from the cap site. Each of these
mutants contains the TATA box (position -26), two GC boxes (positions
-35, -49), and a CCAAT box (position -116) as common cis
elements. Additional upstream elements present in the mutant constructs
are illustrated in Fig. 6.
|
A, the putative
cis elements upstream from the CCAAT box conferred only
slightly higher CAT activity from pICSBP(351)-CAT (4.0%) than those
from pICSBP(151)-CAT (2.9%) in the macrophage cell line. Also shown in
Fig. 7A, both pICSBP(251)-CAT and pICSBP(236)-CAT gave
maximal basal CAT activities (8.2 and 7.9%, respectively) that were
consistently observed to be higher than that of pICSBP(151)-CAT
(2.9%). Interestingly, the activity of pICSBP(179)-CAT, which contains
only the additional pIRE sequence motif upstream of pICSBP(151)-CAT,
was somewhat higher (5.9%) than that of pICSBP(151)-CAT (2.9%),
pICSBP(221)-CAT (3.0%), and pICSBP(351)-CAT (4.0%). These results
suggest that in addition to TATA and CCAAT boxes, the pIRE (position
-155) and GC box (position -227) sequences contribute to optimal
basal expression of the ICSBP gene. The data also suggest that
sequences between positions -351 to -251 and positions -221 to -179
confer an inhibitory effect on basal expression of the ICSBP promoter.
|
-treated EL-4 cells transiently transfected with
pICSBP(351)-CAT, whereas no effect of IFN- was observed with the
pICSBP(151)-CAT construct (37). Those results suggested that sequences
located between positions -151 and -351 were involved in the
induction of ICSBP transcription by IFN-. The pICSBP(351)-CAT
contains several additional cis elements in addition to
those contained within pICSBP(151)-CAT, including a palindromic IRE
(pIRE), an IRE-like motif (GAAA-containing motif resembling the IRE), a
GC box, and an NF-B-like sequence. To address whether the pIRE alone
was sufficient or whether additional upstream sequences were required
for IFN- induction of ICSBP expression, we tested the effect of
IFN- on each construct following transfection into RAW 264.7
macrophages. All constructs, except pICSBP(151)-CAT, which lacks the
pIRE, showed significant up-regulation, with 2- to 5-fold increases in
CAT activity compared with basal levels (Fig. 7, A vs
B). Interestingly, pICSBP(179)-CAT, which contains only the
pIRE (positions -176 to -156) upstream of the CCAAT box, showed
essentially the same level of IFN--inducible CAT activity as that of
pICSBP(236)-CAT (Fig. 7B). This result indicates that the
ICSBP promoter requires only the pIRE in addition to the TATA and CCAAT
boxes for induction of full activity by IFN-.
In contrast to the induction observed in response to IFN- (Fig. 7B), LPS alone failed to induce CAT activity above basal
levels in any of the constructs (Fig. 7C). However, when
stimulated by combined LPS and IFN- treatment, all the constructs
except pICSBP(151)-CAT and pICSBP(221)-CAT showed greater CAT activity
than when treated with IFN- alone (Fig. 7, B vs
D). In addition, the CAT activity of pICSBP(179)-CAT was
almost the same as that of pICSBP(236)-CAT, which showed the maximally
inducible level. This result suggests that even though LPS alone failed
to induce expression of ICSBP promoter in the absence of an authentic
NF-B motif, it somehow increased the ability of IFN- to induce
expression of the ICSBP promoter in RAW 264.7 macrophages.
Effect of ICSBP on its own promoter
ICSBP has been reported to act as a negative
trans-acting factor that suppresses ISRE-containing
promoters (20, 21). The ICSBP promoter also contains IFN-responsive
elements, the pIRE, and an IRE-like motif (38). In addition, ICSBP is
constitutively expressed in cells of the immune system (19, 28). From
our results reported above, pICSBP(179)-CAT showed higher basal CAT
activity than pICSBP(151)-CAT (Fig. 7A), maximal activity
after IFN- induction (Fig. 7B), and near-maximal activity
upon combined stimulation with IFN- plus LPS (Fig. 7D).
To determine whether ICSBP itself plays a role in ICSBP gene
expression, we cotransfected pICSBP(179)-CAT, pICSBP(221)-CAT, or
pICSBP(351)-CAT with an ICSBP expression plasmid (pUE5) or a control
plasmid (pLK440). We also determined the effect of ICSBP overexpression
on a pMHC I-CAT (an MHC class I promoter linked to the CAT reporter
gene). This experiment was performed to confirm the negative regulation
of MHC class I promoter activity by ICSBP that had been reported
previously (3). As shown in Fig. 8,
A and B, ICSBP did not act as a repressor of its
own promoter; instead, it acted as a weak trans-activator of
all three constructs. In the presence of medium alone (Fig. 8A), constitutive expression of ICSBP by the pUE5 construct
resulted in a small, but reproducible, increase in CAT activity above
that observed in cells transfected with the control construct pLK440.
Upon stimulation with IFN-, RAW 264.7 cells transfected with pLK440
exhibited increased CAT activity, which was further augmented by
overexpression of ICSBP (Fig. 8B). These results show that
unlike its effect on MHC I gene expression, ICSBP not only failed to
repress expression of its own promoter, but actually acted as a weak
trans-activator, in part through the pIRE motif contained in
the pICSBP(179)-CAT construct.
|
Support for the possibility that ICSBP can function as a positive
regulator of transcription in macrophages comes from a recent study of
a promoter from the gp91phox phagocyte oxidative burst oxidase
catalytic subunit gene. This study demonstrated that the
gp91phox promoter contains a functional element that binds
ICSBP, IRF-1, and the Ets-like factor PU.1 (41). Sequence analysis of
this element revealed that it contained an Ets core sequence adjacent
to and upstream of an ISRE half-site. A similar functional element was
previously identified in the Ig light chain enhancers and was shown to
bind both IRF-4 and PU.1 (39). PU.1 can bind strongly to these
composite IRF/Ets elements, although IRF-4 binds to DNA only in the
presence of PU.1. Similar findings were later demonstrated using ICSBP
and PU.1 (42). Protein-protein interactions between PU.1 and ICSBP or
IRF-4 have been found to stabilize binding of the IRF proteins to DNA.
We first performed a series of EMSAs to determine whether ICSBP/PU.1
complexes could be formed in RAW 264.7 macrophages, cells that
constitutively express PU.1, and that these complexes specifically
bound to an IRF/Ets composite site. As shown in Fig. 9A, PU.1 and ICSBP present in
macrophage nuclear extracts specifically bound to a DNA probe
containing a single copy of the Ig 3' enhancer composite site
(GGAACTGAAAAC). Abs against PU.1 and ICSBP could supershift the
DNA-protein complexes, whereas Abs against IRF-1 and IRF-2 did not
supershift the complexes (data not shown). Abs that recognize PU.1
blocked the binding of both PU.1 and ICSBP to the DNA, suggesting that
ICSBP could not bind to DNA in the absence of PU.1.
|
could
synergistically activate transcription of a promoter controlled by an
IRF/Ets composite element in macrophages. Therefore, the RAW 264.7
macrophage cell line was transiently transfected with a reporter
plasmid containing the CAT reporter gene under the control of a herpes
simplex virus thymidine kinase promoter ligated downstream of four
tandem copies of the Ig enhancer composite element (plasmid designated
C15) or composite elements in which the IRF (plasmid designated C15m)
binding sites were mutated (GGAACTGAATCA). As shown in Fig. 9B, the C15 reporter plasmid was constitutively expressed in
RAW 264.7 macrophages, whereas mutation of the IRF binding sites
completely abolished this activity. These data demonstrate that an
IRF/Ets composite element can activate transcription in macrophages,
and that the IRF binding site is required for this activation. These
findings are consistent with data obtained in U937 cells using a
reporter gene under the control of the gp91phox IRF/Ets
composite element (41).
We also sought to determine whether ICSBP could function in conjunction
with PU.1 to regulate this promoter. Therefore, RAW 264.7 cells were
cotransfected with the C15 or C15m reporter plasmids and an expression
plasmid encoding ICSBP. A portion of the transfected cells was exposed
to LPS, IFN-, or both agents. As shown in Fig. 9B,
overexpression of ICSBP resulted in more than a 2-fold enhancement of
basal promoter activity. Reporter plasmids in which the IRF binding
sites were mutated lacked basal activity and could not be activated by
overexpression of ICSBP. Furthermore, treatment of transfected cells
with LPS and/or IFN- did not significantly affect the expression of
the C15 reporter plasmid, even in cells that overexpressed ICSBP. In
all cases, the C15 m reporter plasmid was essentially inactive in the
transfected macrophages. Together, these data show that ICSBP can
function as a transcriptional activator in macrophages through specific
interaction with PU.1, but this activation could not be further
modulated by LPS and/or IFN-.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, but not IFN-/ß, in macrophage cell lines (19)
and in thioglycollate-elicited peritoneal macrophages (28, 29).
Induction of ICSBP production at the protein level has also been
demonstrated in murine peritoneal macrophages, and ICSBP has been shown
to localize to the nucleus after treatment of cell with IFN- (29).
ICSBP has been found to be expressed in human cells of the macrophage
and lymphoid lineage and to be similarly induced by IFN- (21).
Functional data using reporter constructs containing IFN-responsive
promoters support a role for ICSBP as a negative transcriptional
regulator in both mouse (20) and human (21) cells. In addition, recent
evidence suggests that ICSBP interacts with IRF-1 and IRF-2 and that
this interaction results in the formation of a complex with an
increased affinity toward the ISRE compared with ICSBP alone (23).
These authors also found that ICSBP has the ability to decrease the
binding of ISGF3 to the ISRE.
We have reported here that LPS and IFN- synergize to induce ICSBP
mRNA, protein, and the formation of ICSBP-DNA complexes in murine
macrophages (Figs. 1 and 9A). This synergy is most clearly
observed at suboptimal levels of IFN- (Fig. 3) and is likely to
result from a rapid and transient burst of ICSBP mRNA production,
peaking approximately 2 h after stimulation with both LPS and
IFN- (Fig. 2). The Lpsd-selective defect in LPS
signaling, present in macrophages from C3H/HeJ macrophages, blocked the
synergistic production of ICSBP mRNA in cells stimulated with both LPS
and IFN-, but had no effect on IFN-mediated signaling (Fig. 4).
Furthermore, synergistic production of ICSBP mRNA was regulated at the
transcriptional level, as indicated by the response of a reporter gene
under the control of a 351-bp fragment of the ICSBP promoter (Fig. 5).
Deletion analysis of the ICSBP promoter identified regions that were
IFN- responsive, regions that mediate the ability of LPS and IFN-
to activate this promoter synergistically, as well as regions that
normally repress ICSBP transcription (Fig. 7). Finally, exogenous
expression of ICSBP, found in previous studies to down-regulate MHC I
gene expression, failed to repress basal or IFN--induced ICSBP
transcription (Fig. 8). This demonstrates that ICSBP can selectively
suppress the expression of IFN-responsive genes. These findings extend
in a significant way our understanding of the regulation of ICSBP by
LPS and IFN- and provide important clues to its role in macrophage
activation.
Cooperation at the level of gene regulation between IFN- and LPS has
been demonstrated with several genes, including, TNF-, iNOS, and
IP-10 (43, 44, 45). We observed similar cooperation for ICSBP at the levels
of protein and steady state mRNA expression as well as in transient
transfection assays using RAW 264.1 cells and the ICSBP-CAT reporter
plasmids. The mechanisms by which the induction of any specific gene is
induced in a synergistic manner are complex and depend on the presence
of various cis-acting elements within the promoter region
and their coordinate engagement by DNA binding proteins (46). For
example, Gao et al. (7) have recently proposed a model to explain the
limited activation of iNOS gene expression when stimulated by LPS vs
LPS plus IFN-. In their model, IFN- generated phosphorylated
Stat1 homodimers preferentially, and these engage a GAS element that
has been shown previously to be essential for iNOS gene expression
(47). IFN- can also concurrently elicit the induction of other IRF
family members, including IRF-1, which was also shown previously to be
essential for the induction of this gene (48, 49). The additional
presence of LPS generates additional signaling molecules (e.g., NF-B
and IRF-1) as well as IFN-/ß, which, in turn, acts back on the
macrophage to induce additional activated Stat1 (7). In concert with
additional, constitutively produced octamer-binding factors, coordinate
engagement of these six cis-acting elements results in
optimal transcription. A similar model has been proposed by Ohmori et
al. (45) for the regulation of IP-10 in murine macrophages. Moreover,
it is likely that the IFN-responsive elements within the ICSBP promoter
are not shared by LPS-responsive elements. This conclusion comes from
studies of Lpsd macrophages, which exhibited normal
production of ICSBP mRNA following stimulation with IFN- alone and
did not exhibit synergistic activation following treatment with both
LPS and IFN-.
The 5' regulatory region of the murine ICSBP gene has been
characterized (38), and the sequence between positions -175 and -155
identified as an IFN- response region, called the pIRE (see Footnote
5). In IFN--activated EL-4 T cells, activation of ICSBP has been
associated with activation of Stat1, which has been shown to bind to
the pIRE of both IRF-1 and ICSBP promoters (37). GC box and IRE-like
sequences were also identified at positions -222 to -227 and -217 to
-198, respectively (38). In this study, we generated deletion mutants
(Figs. 6 and 7) to explore the contributions of these various
cis-acting elements to transcriptional activation of ICSBP
by IFN- alone, LPS alone, or both activators. In promoter mutants
containing only the pIRE (pICSBP(179)-CAT), this element was sufficient
to mediate optimal basal and IFN--induced transcription. LPS alone
had no stimulatory effect above basal levels. However, the combination
of LPS and IFN- resulted in significantly increased CAT activity in
four of five reporter plasmids. Our data contrast with previous studies
conducted in the murine EL-4 T-cell line, where pICSBP(151)-CAT
resulted in basal CAT activity somewhat higher than that induced by
pICSBP(351)-CAT (37). This suggests that the transcriptional factors
involved in basal expression of the ICSBP promoter may differ between T
cells and macrophages. The finding that synergy is observed in
constructs that lack the NF-B-like motif (which is present in the
pICSBP(351)-CAT construct) suggests that LPS-induced
trans-acting factors apart from NF-B contribute to the
synergistic activation of such constructs in the presence of IFN-
plus LPS. Alternatively, LPS-induced NF-B may interact directly with
other transcription factors via protein-protein interaction without
direct binding to DNA. Thus, the absence of an NF-B binding motif
would not affect its ability to contribute to functional synergy via
protein-protein interaction. It is interesting to note that the
pICSBP(221)-CAT and pICSBP(351)-CAT constructs showed significantly
diminished responses compared with the minimal pIRE-containing
construct. Thus, engagement of an incomplete repertoire of
cis-acting elements can exert an inhibitory effect on
transcription. Interestingly, one construct (pICSBP(221)-CAT) failed to
exhibit LPS responsiveness in the presence or the absence of IFN-,
although both shorter (pICSBP(179)-CAT) and longer (pICSBP(236)-CAT)
constructs exhibited increased CAT activity in the presence of both LPS
and IFN-. The molecular basis for this observation remains unclear
and is under investigation.
Finally, our data show that ICSBP can function in macrophages as a
transcriptional activator of its own promoter (Fig. 8), and of a
synthetic PU.1-dependent promoter (C15) through specific interaction
with an IRF/Ets composite element (Fig. 9). Overexpression of ICSBP
protein could further enhance transcription of the ICSBP promoter by
LPS and IFN-. In contrast, enhanced activity of the C15 promoter by
ICSBP could not be further modulated by LPS and/or IFN-. Thus, we
have shown that transcriptional activity of the ICSBP promoter
generally correlates with ICSBP protein levels, although transcription
of a promoter regulated by ICSBP/PU.1 complexes did not. Therefore, the
ability of LPS and IFN- to increase ICSBP protein levels
synergistically in macrophages may have a selective effect on different
ICSBP-dependent genes. Although stimulation with LPS plus IFN-,
which induced endogenous ICSBP expression in RAW 264.7 macrophages,
does not affect C15 transcription (Fig. 9), overexpression of ICSBP
encoded by a transfected plasmid increased C15 transcription
approximately 2-fold. This most likely results from the much higher
levels of ICSBP protein present in cells transfected with the ICSBP
expression plasmid than those in cells stimulated with LPS plus
IFN-. Moreover, these high ICSBP levels are sustained in the ICSBP
transfectants due to the strong CMV promoter in the expression plasmid.
In contrast to the observation that endogenous ICSBP mRNA is rapidly,
but transiently, expressed in macrophages treated with LPS plus IFN-
(Fig. 2 and data not shown), much higher levels of ICSBP are generated
by transient transfection compared with LPS plus IFN- stimulation
over the 24-h course of the experiment presented in Fig. 9. Lastly, the
C15 promoter is likely to be less sensitive to changes in ICSBP levels
compared with native promoters because the C15 promoter contains only
an IRF/Ets-responsive element and lacks additional LPS- and
IFN-responsive regulatory elements that would be present in native
promoters.
Together, these results provide some of the first demonstrations that
ICSBP is not a universal trans-repressor. Under conditions
where MHC I gene expression is repressed by constitutive expression of
ICSBP, ICSBP actually acted as a weak trans-activator in
either the absence or the presence of IFN-. More recent studies
suggest that this may also be the case for other macrophage genes.
ICSBP has been found to stimulate transcription of the IL-12 p40
promoter via interaction with Ets-2 (K. Ozato, in preparation). In
combination with PU.1 and IRF-1, ICSBP has been demonstrated to
activate the gp91phox IRF/Ets composite element (41). The
apparent ability of ICSBP to function as a dichotomous regulator of
transcription is similar to the characteristics of other IRFs,
including IRF-2, IRF-3, and IRF-4/Pip (Ref. 9 and references therein).
At this juncture, the role of ICSBP in macrophage activation can be
best assessed from studies using ICSBP knockout mice. The failure of
macrophages obtained from these mice to control intracellular parasites
is consistent with their failure to elicit the production of IL-12, and
consequently IFN- (26). Specifically, macrophages from these
ICSBP-deficient mice fail to express the p40 subunit of IL-12 (26).
Together, these data suggest that ICSBP plays a critical role in the
development of the fully activated macrophage. A role for ICSBP in the
control of myeloid leukemias has also been proposed. Schmidt et al.
recently reported a lack of ICSBP transcripts in human myeloid
leukemias (50). This finding is consistent with an earlier report that
ICSBP-deficient mice spontaneously develop a chronic myelogenous
leukemia-like syndrome that is highly similar to the human disease
(25). Our understanding of the contribution of ICSBP to both innate
immunity and the development of myeloid leukemia will depend in part on
defining the molecular basis for ICSBP gene expression and its capacity
to serve as a positive and a negative regulator of IFN-responsive
genes.
|
Footnotes |
|---|
2 W.K. and A.D.P contributed equally to the work presented in this report.
3 Address correspondence and reprint requests to Dr. Stefanie N. Vogel, Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road., Bethesda, MD 20814. E-mail address:
4 Abbreviations used in this paper: ICS, IFN consensus sequence; iNOS, inducible NO synthase; ISRE, IFN-stimulated response element; IRF, IFN regulatory factor; ISGF-3, IFN-stimulated gene factor-3; ICSBP, ICS binding protein; CAT, chloramphenicol acetyltransferase; IRE, IFN response element; pIRE, palindromic IRE.
5 Although originally referred to as pIRE and IRE-like motifs (38), the present nomenclature would be -activated sequence and ISRE-like motifs, respectively.
Received for publication December 15, 1998. Accepted for publication March 30, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
. Proc. Natl. Acad. Sci. USA 80:3782./ß mediates the LPS-induced activation of transcription factor Stat1 in mouse macrophages: pivotal role of Stat1 in induction of the inducible nitric oxide synthase gene. J. Immunol. 161:4803.-Interferon-induced transcription of HLA and metallothionein genes containing homologous upstream sequences. Nature 314:637.[Medline]
in a cell-free system. Science 257:813., consists of multiple interaction polypeptide chains. Proc. Natl. Acad. Sci. USA 87:8555.-induced transcriptional activator, define a gene family involved in signal transduction. Proc. Natl. Acad. Sci. USA 89:7840.. Proc. Natl. Acad. Sci. USA 89:7836.-regulated protein that binds the interferon inducible enhancer element of major histocompatability complex class I genes. Proc. Natl. Acad. Sci. USA 87:3743. and down-regulation by IFN-, dexamethasone, and protein kinase inhibitors. J. Immunol. 148:801.[Abstract]
-induces nuclear expression of IFN consensus sequence binding protein in murine peritoneal macrophages. J. Immunol. 152:2270.[Abstract]
interferon-responsive element, to which an ISGF3 subunit (or similar) molecule binds. Mol. Cell. Biol. 13:3951. 3' enhancer activity. Mol. Cell. Biol. 12:368.-interferon on cachectin expression by mononuclear phagocytes: reversal of the Lpsd (endotoxin resistance) phenotype. J. Exp. Med. 164:1791. and lipopolysaccharide. J. Biol. Chem. 268:1908.B sequence motifs controls IFN- and lipopolysaccharide-stimulated transcription from the murine IP-10 promoter. J. Biol. Chem. 268:6677.-activated site (GAS) is necessary for full expression of the mouse iNOS gene in response to interferon- and lipopolysaccharide. J. Biol. Chem. 272:1226.This article has been cited by other articles:
|
|
 |
|
  A. la Sala, J. He, L. Laricchia-Robbio, S. Gorini, A. Iwasaki, M. Braun, G. S. Yap, A. Sher, K. Ozato, and B. Kelsall Cholera toxin inhibits IL-12 production and CD8{alpha}+ dendritic cell differentiation by cAMP-mediated inhibition of IRF8 function J. Exp. Med., June 8, 2009; 206(6): 1227 - 1235. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Koenigsmann, C. Rudolph, S. Sander, O. Kershaw, A. D. Gruber, L. Bullinger, B. Schlegelberger, and D. Carstanjen Nf1 haploinsufficiency and Icsbp deficiency synergize in the development of leukemias Blood, May 7, 2009; 113(19): 4690 - 4701. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. J. Kong, D. E. Anderson, C. H. Lee, M. K. Jang, T. Tamura, P. Tailor, H. K. Cho, J. Cheong, H. Xiong, H. C. Morse III, et al. Cutting Edge: Autoantigen Ro52 Is an Interferon Inducible E3 Ligase That Ubiquitinates IRF-8 and Enhances Cytokine Expression in Macrophages J. Immunol., July 1, 2007; 179(1): 26 - 30. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Schroder, M. Lichtinger, K. M. Irvine, K. Brion, A. Trieu, I. L. Ross, T. Ravasi, K. J. Stacey, M. Rehli, D. A. Hume, et al. PU.1 and ICSBP control constitutive and IFN-{gamma}-regulated Tlr9 gene expression in mouse macrophages J. Leukoc. Biol., June 1, 2007; 81(6): 1577 - 1590. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Yang, Y. Chu, Y. Wang, R. Zhang, and S. Xiong Targeted in vivo expression of IFN-{gamma}-inducible protein 10 induces specific antitumor activity J. Leukoc. Biol., December 1, 2006; 80(6): 1434 - 1444. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Zhu, M. S. Chang, R. C. Hsueh, R. Taussig, K. D. Smith, M. I. Simon, and S. Choi Dual Ligand Stimulation of RAW 264.7 Cells Uncovers Feedback Mechanisms That Regulate TLR-Mediated Gene Expression J. Immunol., October 1, 2006; 177(7): 4299 - 4310. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Lehtonen, V. Veckman, T. Nikula, R. Lahesmaa, L. Kinnunen, S. Matikainen, and I. Julkunen Differential Expression of IFN Regulatory Factor 4 Gene in Human Monocyte-Derived Dendritic Cells and Macrophages J. Immunol., November 15, 2005; 175(10): 6570 - 6579. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Xiong, H. Li, H. J. Kong, Y. Chen, J. Zhao, S. Xiong, B. Huang, H. Gu, L. Mayer, K. Ozato, et al. Ubiquitin-dependent Degradation of Interferon Regulatory Factor-8 Mediated by Cbl Down-regulates Interleukin-12 Expression J. Biol. Chem., June 24, 2005; 280(25): 23531 - 23539. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Zhu, G. Saberwal, Y. Lu, L. C. Platanias, and E. A. Eklund The Interferon Consensus Sequence-binding Protein Activates Transcription of the Gene Encoding Neurofibromin 1 J. Biol. Chem., December 3, 2004; 279(49): 50874 - 50885. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Alter-Koltunoff, S. Ehrlich, N. Dror, A. Azriel, M. Eilers, H. Hauser, H. Bowen, C. H. Barton, T. Tamura, K. Ozato, et al. Nramp1-mediated Innate Resistance to Intraphagosomal Pathogens Is Regulated by IRF-8, PU.1, and Miz-1 J. Biol. Chem., November 7, 2003; 278(45): 44025 - 44032. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Kuwata, C. Gongora, Y. Kanno, K. Sakaguchi, T. Tamura, T. Kanno, V. Basrur, R. Martinez, E. Appella, T. Golub, et al. Gamma Interferon Triggers Interaction between ICSBP (IRF-8) and TEL, Recruiting the Histone Deacetylase HDAC3 to the Interferon-Responsive Element Mol. Cell. Biol., November 1, 2002; 22(21): 7439 - 7448. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. S. Kim, E. J. Park, E.-h. Joe, and I. Jou JAK-STAT Signaling Mediates Gangliosides-induced Inflammatory Responses in Brain Microglial Cells J. Biol. Chem., October 18, 2002; 277(43): 40594 - 40601. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Tsujimura, T. Nagamura-Inoue, T. Tamura, and K. Ozato IFN Consensus Sequence Binding Protein/IFN Regulatory Factor-8 Guides Bone Marrow Progenitor Cells Toward the Macrophage Lineage J. Immunol., August 1, 2002; 169(3): 1261 - 1269. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Kallies, F. Rosenbauer, M. Scheller, K.-P. Knobeloch, and I. Horak Accumulation of c-Cbl and rapid termination of colony-stimulating factor 1 receptor signaling in interferon consensus sequence binding protein-deficient bone marrow-derived macrophages Blood, May 1, 2002; 99(9): 3213 - 3219. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. A. Doughty, K. B. Nguyen, J. E. Durbin, and C. A. Biron A Role for IFN-{{alpha}}{{beta}} in Virus Infection-Induced Sensitization to Endotoxin J. Immunol., February 15, 2001; 166(4): 2658 - 2664. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Giroux and A. Descoteaux Cyclooxygenase-2 Expression in Macrophages: Modulation by Protein Kinase C-{alpha} J. Immunol., October 1, 2000; 165(7): 3985 - 3991. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. A. Cramer, S. L. Nelson, and M. J. Klemsz Synergistic Induction of the Tap-1 Gene by IFN-{gamma} and Lipopolysaccharide in Macrophages Is Regulated by STAT1 J. Immunol., September 15, 2000; 165(6): 3190 - 3197. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y.-M. Kim, J. Y. Im, S. H. Han, H. S. Kang, and I. Choi IFN-{gamma} Up-Regulates IL-18 Gene Expression Via IFN Consensus Sequence-Binding Protein and Activator Protein-1 Elements in Macrophages J. Immunol., September 15, 2000; 165(6): 3198 - 3205. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I-M. Wang, C. Contursi, A. Masumi, X. Ma, G. Trinchieri, and K. Ozato An IFN-{gamma}-Inducible Transcription Factor, IFN Consensus Sequence Binding Protein (ICSBP), Stimulates IL-12 p40 Expression in Macrophages J. Immunol., July 1, 2000; 165(1): 271 - 279. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Hein, V. A. J. Kempf, J. Diebold, N. Bucheler, S. Preger, I. Horak, A. Sing, U. Kramer, and I. B. Autenrieth Interferon Consensus Sequence Binding Protein Confers Resistance against Yersinia enterocolitica Infect. Immun., March 1, 2000; 68(3): 1408 - 1417. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Meraro, S. Hashmueli, B. Koren, A. Azriel, A. Oumard, S. Kirchhoff, H. Hauser, S. Nagulapalli, M. L. Atchison, and B.-Z. Levi Protein-Protein and DNA-Protein Interactions Affect the Activity of Lymphoid-Specific IFN Regulatory Factors J. Immunol., December 15, 1999; 163(12): 6468 - 6478. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Marecki, M. L. Atchison, and M. J. Fenton Differential Expression and Distinct Functions of IFN Regulatory Factor 4 and IFN Consensus Sequence Binding Protein in Macrophages J. Immunol., September 1, 1999; 163(5): 2713 - 2722. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. A. Salkowski, K. Kopydlowski, J. Blanco, M. J. Cody, R. McNally, and S. N. Vogel IL-12 Is Dysregulated in Macrophages from IRF-1 and IRF-2 Knockout Mice J. Immunol., August 1, 1999; 163(3): 1529 - 1536. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Cohen, A. Azriel, T. Cohen, D. Meraro, S. Hashmueli, D. Bech-Otschir, R. Kraft, W. Dubiel, and B.-Z. Levi Interaction between Interferon Consensus Sequence-binding Protein and COP9/Signalosome Subunit CSN2 (Trip15). A POSSIBLE LINK BETWEEN INTERFERON REGULATORY FACTOR SIGNALING AND THE COP9/SIGNALOSOME J. Biol. Chem., December 8, 2000; 275(50): 39081 - 39089. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
