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and Lipopolysaccharide in Macrophages Is Regulated by STAT11
Department of Microbiology and Immunology, Indiana University School of Medicine, and Walther Cancer Institute, Indianapolis, IN 46202
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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activation site element is critical for the rapid
induction of the promoter by IFN- following transfection into the
human macrophage cell line THP-1. Furthermore, activation of STAT1
binding to this site was important for the synergistic response seen
following the stimulation with both IFN- and LPS. Mutation of an
IFN-stimulated regulatory element that binds IFN regulatory factor 1
appeared to enhance the response to IFN- and LPS. These data show
that STAT1 is necessary for the activation of Tap-1 gene expression in
APCs and initiation of cellular immune responses. Furthermore, our data
suggest that bacterial products such as LPS may enhance cellular immune
responses through augmenting the ability of STAT1 to regulate
IFN--inducible genes. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have focused on how the Tap-1 and Tap-2 genes are regulated in
professional APCs, the first step in activating a cellular immune
response. Our results have shown that in the human macrophage cell line
THP-1, the Tap-1 gene is expressed at almost undetectable levels, while
the Tap-2 gene is expressed at moderate levels (3).
Following stimulation with IFN-, the Tap-1 gene is induced at the
mRNA and protein levels. We have also shown by nuclear run-on analysis
that the Tap-1 gene is regulated at the level of transcription in this
cell line following stimulation with IFN-. Several other reports
have shown that increases in Tap-1 mRNA and protein levels can be seen
in HeLa cells (4), human vascular endothelial cells
(5), and human keratinocytes (6) following
stimulation with IFN-. In addition, we have shown that LPS altered
the kinetics and increased the overall fold induction of
IFN--induced Tap-1 gene expression in the human macrophage cell line
THP-1 (3). This was seen at the mRNA, transcription, and
protein levels in THP-1 cells, but the synergistic LPS effect was not
seen at any level in the nonhemopoietic cell line HeLa
(3). This suggested that the overall regulation of the
Tap-1 gene may be different in professional APCs such as macrophages,
as compared with other cell types.
Based on changing levels of TAP-1 mRNA expression seen by Northern
analysis, several groups have begun studies on the Tap-1 promoter.
Interestingly, the Tap-1 gene is regulated by a bidirectional promoter
that it shares with a gene important for the processing of Ags, low
molecular mass polypeptide 2
(LMP2)4
(7, 8, 9). In humans, the region between these two genes that
are transcribed in opposite directions is 
593 bp (7).
Studies on this regulatory region have shown that it can regulate
expression of a reporter gene in both the Tap-1 and LMP2 directions
(4, 8). Within the Tap-1/LMP2 bidirectional promoter, four
elements have been mapped and studied. These are Sp-1 and NF-
B
sites, and a combination IFN-stimulated regulatory element (ISRE) and
IFN- activation site (GAS) element. The NF-B site has been shown
to be important for the induction of promoter activity following
transient transfection and stimulation with TNF-
(4, 8). The data regarding the regulation of the Tap-1 promoter by
IFN- in nonimmune cells such as HeLa cells have been conflicting
(4, 9). The published reports appear to disagree about
whether the GAS site, which binds STAT1, or the ISRE site, which binds
IFN regulatory factor 1 (IRF-1), is more important for the response to
stimulation with IFN-. No reports have shown which factor is
involved in the response to IFN- in cells of the immune system.
Although other groups have studied the promoter region, no data have
been published showing that the Tap-1 or LMP2 genes are regulated at
the level of transcription in other cell types. Our studies using
nuclear run-on assays have shown that the Tap-1 gene is regulated
transcriptionally in THP-1 cells. We have also shown that the synergy
between IFN- and LPS is only seen in this macrophage cell line.
Thus, studies on the regulation of the Tap-1/LMP2 bidirectional
promoter in THP-1 cells would provide important information on how this
gene is regulated during the initiation of an immune response.
In this study, we have investigated the regulation of this promoter in
the human macrophage cell line, THP-1. Our results show that in this
macrophage line, STAT1 is the critical transcription factor that
initiates Tap-1 gene expression following stimulation with IFN-.
Furthermore, the ability of LPS to synergize with IFN- also works
through the STAT1 binding site. These data suggest that LPS and other
bacterial products may enhance presentation of Ags through augmenting
IFN--regulated gene expression in macrophages.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The human macrophage cell line, THP-1, was cultured in RPMI 1640
(BioWhittaker, Walkersville, MD) media with 10% Fetalclone I (HyClone
Laboratories, Logan, UT) without antibiotics at 37°C with 7.5%
CO2. THP-1 cells were maintained in cell culture
between 5 x 105 and 1.5 x
106 cells/ml. HeLa cells were maintained in DMEM
(BioWhittaker) with 5% Fetalclone I and penicillin/streptomycin. Human
IFN- was purchased from Roche Molecular Biochemicals (Indianapolis,
IN). LPS (Escherichia coli serotype 055:B5) was purchased
from Sigma (St. Louis, MO).
Reporter plasmids
The Tap-1/LMP2 bidirectional promoter was cloned from genomic DNA isolated from THP-1 cells using PCR. The full-length promoter fragment was cloned into KS+ and sequenced. The promoter was cloned directionally into the luciferase reporter plasmid pXP2 (10). Deletions were generated by PCR, using a series of 5' oligonucleotides and the same 3' oligonucleotide used for the generation of the full-length promoter. Mutations of individual binding sites were made using overlap extension PCR, as described (11). All deletion and mutation inserts were sequenced before insertion into the luciferase reporter plasmid, pXP2.
To generate the p(I/G)3 Luc reporter plasmid, a minimal thymidine kinase (TK) promoter was first cloned into pXP2 to generate the plasmid, pBLuc. Next, oligonucleotides representing the combination ISRE/GAS element from the Tap-1 promoter were ligated into pBLuc, and individual clones were sequenced. p(I/G)3 Luc contains three oligonucleotides ligated in frame in the pBLuc plasmid.
Transient transfections
THP-1 cells were transfected using DEAE-dextran. Briefly, cells were washed twice in serum-free RPMI 1640 and concentrated to 1 x 107 cells/ml. At the same time, the reporter plasmids were incubated in serum-free media and a final concentration of 100 mM Tris, pH 7.4, for 5 min. Following the addition of DEAE-dextran to a final concentration of 75 µg/ml and incubation for 15 min, 1 ml of cells was added for each transfection reaction. The transfection reactions were incubated for 40 min at 37°C with 7.5% CO2, washed once, and resuspended in 10 ml of RPMI 1640 containing 10% Fetalclone I. The transfected cells were allowed to recover for 18 h, followed by stimulation for the indicated times and conditions. Transfections were harvested, washed once with cold PBS, and resuspended in lysis buffer. Luciferase activity was measured using standard procedures on a Lumat LB 9501 luminator (Berthold Systems, Pittsburgh, PA). HeLa cells were transfected with Lipofectin (Life Technologies, Gaithersburg, MD), as previously described (12).
Nuclear extract preparation and band-shift analysis
Nuclear extracts were prepared as previously described (11). Briefly, pellets of THP-1 cells were resuspended in TKM buffer (50 mM Tris, pH 8, 50 mM KCl, 15 mM MgCl2), followed by the addition of an equal volume of 0.3 M sucrose. After adding Nonidet P-40 to a final concentration of 0.1%, the tubes were inverted 10 times and the nuclei were pelleted. The nuclei were resuspended in nuclear extraction buffer (20 mM HEPES, pH 7.9, 5% glycerol, 1.5 mM MgCl2, 0.2 mM EDTA, 0.42 M NaCl plus the protease inhibitors, pepstatin, aprotinin, leupeptin, and Pefabloc). After incubation on ice for 30 min, the chromatin was pelleted and the nuclear proteins were quantitated. Whole cell extracts were prepared by lysing cell pellets in cell lysis solution 1 (25 mM Tris, pH 8, 75 mM NaCl, 0.05 mM EDTA, 0.5% Nonidet P-40, 1 mM Na3VO4, 5 mM NaF, 5 mM ß-glycerol-phosphate, 1 mM DTT, 1 mM Pefabloc, 2 µg/ml aprotinin, and 2 µg/ml pepstatin A). Following incubation on ice for 15 min, the lysed cells were centrifuged for 15 min at 40°C. The supernatants were removed and the proteins were quantitated by the method of Lowery (Bio-Rad, Hercules, CA).
Band-shift analysis was performed as described (11, 12). Briefly, binding reactions were performed in a final volume of 20 µl containing 1x binding buffer (20 mM HEPES, pH 7.9, 75 mM KCl, 1 mM DTT, 0.5 mM EDTA), 2 µg of poly(dI/dC), and indicated amounts of nuclear extracts. For binding reactions with the combination I/G element, salmon sperm was substituted for poly(dI/dC). Reactions were incubated for 15 min at room temperature, followed by electrophoresis at 4°C on a 6% acrylamide gel containing 5% glycerol and 0.25x TBE. For supershift experiments, following the initial binding reaction, appropriate Abs were added and incubated for the indicated times. All Abs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Band-shift gels were dried and bands were visualized by autoradiography.
Oligonucleotides
The following oligonucleotides were used for transfection and
band-shift analyses. The I/G oligonucleotides were cloned into the
reporter plasmid, pBLuc, to generate p(I/G)3 Luc, as
described above. All pairs of oligonucleotides were cloned into
KS+ before use as probes for band-shift analysis.
The sequence of only the top strand (a) for each pair of
oligonucleotides is shown. Lower case letters at the 5' end of an
oligonucleotide represent additional bases added to assist in the
cloning of these oligonucleotides. Lower case letters within
the oligonucleotides represent bases that were mutated.
I/G, 5'-gatcGGCCGCTTTCGATTTCGCTTTCCCCTAAATGGCTGAG; mI/G,
5'-gatcGGCCGCTTTCGAcacCGCTTTCCCCTAAATGGCTGAG; I/mG,
5'-gatcGGCCGCTTTCGATTTCGCTTTCCCCaggATGGCTGAG; mI/mG,
5'-gatcGGCCGCTTTCGAcacCGCTTTCCCCaggATGGCTGAG; GAS,
5'-TTCCCCTAAATGGCTGAG; ISRE, 5'-GCTTTCGATTTCGCTTTC; NF-B,
5'-TTCCTGGGACTTTCCGAG.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have previously shown by nuclear run-on analysis that the
induction of the Tap-1 gene by IFN- in the human macrophage cell
line THP-1 is controlled at the level of transcription
(3). To investigate which cis-acting elements
are critical for this induction, we isolated the human Tap-1/LMP2
bidirectional promoter from THP-1 genomic DNA. In addition, we created
three deletions of the promoter. This series of luciferase reporter
plasmids were transfected into THP-1 cells, followed by stimulation
with IFN-, LPS, or both for 24 h. The results showed that
deletion of 370 bp (pLTPb) from the LMP2 end of the promoter had no
effect on the induction by any stimulation (Fig. 1
). Further deletion of the ISRE/GAS
region (pLTPc), which contains potential DNA binding sites for both
IRF-1 and STAT1, resulted in the loss of ability of the reporter
plasmid to respond to stimulation.
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). Results of transfection of these mutant promoters
into THP-1 cells showed that loss of the GAS binding site (pLTPmG)
almost completely abrogated the ability of this promoter to respond to
IFN-. However, loss of the ISRE binding site (pLTPmI) had no effect.
In fact, loss of this binding site resulted in a higher fold induction
following stimulation with IFN-. These data show that in
macrophages, the ISRE/GAS region is required for the induction of Tap-1
gene expression. Furthermore, our data show that in macrophages, the
GAS element is more important than the ISRE element for the initial
induction of Tap-1 gene expression following stimulation with
IFN-. Binding of STAT1 or IRF-1 to the GAS and ISRE regions of the Tap-1 promoter
Our transfection data suggested that the GAS element was critical
for induction of the Tap-1 promoter. This would imply that STAT1 was
the trans-acting factor necessary for the IFN--induced
expression of Tap-1 in macrophages. To show that STAT1-binding activity
was induced in THP-1 cells, and that the mutations we used for the
transient transfection experiments only affected STAT1 binding, we used
band-shift analysis to determine which trans-acting factors
were bound to the combination element. Previous studies had shown that
in nuclear extracts from HeLa cells stimulated with IFN- binding of
both STAT1 and IRF-1 to the human Tap-1 promoter could be detected by
band-shift analysis. In a similar fashion, our results showed that both
STAT1 and IRF-1 binding could be detected in nuclear extracts from
THP-1 cells stimulated with IFN- for 4 h (Fig. 2). Using oligonucleotide probes for
either the GAS or the ISRE site, supershift analysis using Abs to each
of these factors showed that STAT1 bound to the GAS site, and IRF-1
bound to the ISRE site, but neither protein bound to the other
site.
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).
Likewise, IRF-1 binding to the isolated ISRE site could only be
competed by the wild-type oligonucleotide or when the GAS site was
mutated (ISRE/mGAS), but not when the ISRE site was mutated
(mISRE/GAS). These data show that both STAT1 and IRF-1 can be induced
in THP-1 cells, and that they can bind independently to the combination
element found in the Tap-1 promoter. Mutation of the GAS element, which
was critical for the induction of Tap-1 promoter activity in THP-1
cells by IFN-, only prevented binding of STAT1 and not IRF-1.
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Band-shift results have shown that both STAT and IRF-1 can be
induced in THP-1 cells following stimulation with IFN- for 4 h.
Because our transfection results implicated STAT1, we used nuclear
extracts made from THP-1 cells stimulated with IFN- over a time
course. STAT1-binding activity to the isolated GAS element was induced
in THP-1 cells within 5 min of stimulation with IFN-, and could be
detected over the entire 24 h (Fig. 4). In comparison, IRF-1 binding to the
ISRE element was not detected until these cells had been stimulated for
2 h (Fig. 4). We next asked whether these proteins would also bind
the combination ISRE/GAS element. Using whole cell extracts, which gave
a lower level of background with this probe, STAT1 clearly bound to the
combination ISRE/GAS element (Fig. 5).
Interestingly, there was a lower level of IRF-1-binding activity to the
combination element as compared with the isolated ISRE site (Fig. 5).
Based on both the transfection and band-shift analyses, our data
suggest that induction of STAT1 binding to the Tap-1 promoter is
critical for the initial IFN--induced expression of this gene in
macrophages.
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and LPS following transfection of the Tap-1
promoter
In addition to showing the importance of STAT1 in the initial
induction of Tap-1 gene expression by IFN-, we wanted to show how
LPS was able to synergistically increase the expression of the Tap-1
gene in THP-1 cells. In our initial transfections of the Tap-1
promoter, stimulation with both IFN- and LPS for 24 h showed
approximately the same fold induction as stimulation with IFN- alone
(Fig. 1). One possible explanation is that stimulation for 24 h
following transfection missed a critical time point of induction for us
to see the synergistic effect of LPS. To test this, we transfected the
full-length Tap-1 promoter luciferase reporter plasmid pLTP into THP-1
cells, and stimulated for varying amounts of time. The results showed
that maximal induction of reporter gene activity was seen within 4
h of stimulation with IFN- alone (Fig. 6). This increase was sustained through
8 h of stimulation, but declined by 24 h. More importantly,
this time course experiment also showed a dramatic synergistic increase
in reporter gene activity following stimulation with both IFN- and
LPS. The maximal fold induction was seen after 4 h, and these
levels declined to the level following stimulation with IFN- alone
after 24 h. These data show that LPS can synergistically increase
the activity of the Tap-1 promoter following transfection into THP-1
cells. This is consistent with our earlier findings showing that
stimulation of this macrophage cell line with both IFN- and LPS
synergistically increases the transcription, mRNA, and protein levels
of the Tap-1 gene, as compared with stimulation with IFN- alone.
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The time course stimulations following transfection of the
full-length Tap-1 promoter showed that IFN- and LPS induced maximal
reporter gene activity within 4 h of stimulation. Therefore, we
transfected our mutant I and mutant G Tap-1 promoter reporter plasmids
and stimulated the cells over the same time course. The data from these
experiments showed that mutation of the GAS site significantly reduced
the overall fold induction throughout the time course (Fig. 7, left). However, IFN- and
LPS stimulation of cells transfected with the GAS mutant still showed a
significant increase over the first 4–8 h of stimulation as compared
with stimulation with IFN- alone. These levels were reduced to the
same levels as stimulation with IFN- alone after 24 h, as was
the case for the wild-type promoter. Mutation of the ISRE had no effect
on the response to stimulation with IFN- alone throughout the time
course (Fig. 7, right). Interestingly, stimulation with
IFN- and LPS for 4 or 8 h revealed a significantly larger
increase in the fold induction of reporter gene activity, as compared
with the wild-type Tap-1 promoter. Finally, transfection of a reporter
plasmid with mutations in both the GAS and ISRE sites showed background
levels of induction (data not shown). These data show that the GAS site
is critical for the initial induction of Tap-1 promoter activity
following stimulation with IFN-, and stimulation with IFN- plus
LPS. Mutation of the ISRE site suggests that this element may only play
a role late in the response of the Tap-1 promoter to IFN-.
Furthermore, it appears to play a negative role early in the reponse to
IFN- plus LPS. Mutation of both sites completed abrogated any
induction of reporter gene activity following transfection into THP-1
cells.
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Our data suggest that the ISRE/GAS element is important for the
induction of Tap-1 promoter activity in THP-1 cells stimulated with
IFN- alone or IFN- plus LPS. Because the promoter also contains a
NF-B site, which could be important for the LPS response, we mutated
this site and transfected THP-1 cells. The data showed that loss of the
NF-B site had no effect on the Tap-1 promoter following stimulation
with IFN- alone or the combination of IFN- and LPS (data not
shown). To further analyze the isolated ISRE/GAS element, we created a
reporter plasmid that contains a minimal TK promoter and three copies
of this element. Transfection of this plasmid, p(I/G)3 Luc,
into THP-1 cells responded to stimulation with IFN-. The addition of
LPS with the IFN- resulted in a further increase in luciferase
activity after 4 h of stimulation. This induction was reduced to
IFN- levels after 24 h, which was comparable with our results
with the full-length Tap-1 promoter (Fig. 8). We also transfected these plasmids
into HeLa cells. Although both responded to stimulation with IFN-,
neither plasmid showed an increase when LPS was added with the IFN-
(data not shown). These data show that the GAS element is necessary for
the response of the Tap-1 promoter to stimulation with either IFN-
alone or the combination of IFN- and LPS. This response is specific
to the macrophage line, THP-1, and is consistent with our previous
findings (3).
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and LPS
To begin to understand how the combination of IFN- and LPS
synergistically increased Tap-1 promoter activity, we isolated nuclear
extracts from THP-1 cells stimulated for 30 min with IFN- alone, LPS
alone, or the combination of both IFN- and LPS. Band-shift analysis
using the isolated GAS element showed that more STAT1-binding activity
was found in the extracts from the THP-1 cells stimulated with both
IFN- and LPS, as compared with stimulation with IFN- alone (Fig. 9). These data suggest that LPS can
augment the amount of STAT1 seen in the nucleus following stimulation
with IFN-.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in macrophages.
Mutation of these two sites showed that the GAS site was necessary for
the IFN- response. Band-shift analysis showed that STAT1 bound to
this GAS site in macrophages. A synergistic increase in reporter gene
activity was seen following stimulation of the Tap-1
promoter-transfected cells with both IFN- and LPS. Interestingly,
the STAT1-binding GAS site was critical for this synergistic response
to both stimuli. The increase in reporter gene activity could be
mimicked using an isolated ISRE/GAS element ligated into a minimal TK
promoter luciferase reporter plasmid. Increased binding of STAT1 to the
GAS element was also seen following stimulation with both IFN- and
LPS, as compared with stimulation with IFN- alone. These results
suggest that the expression of the Tap-1 gene was controlled in
macrophages by STAT1. More importantly, the synergistic increase in
Tap-1 gene expression in macrophages following stimulation with both
IFN- and LPS requires STAT1. This suggests that bacterial products
such as LPS may enhance the ability of macrophages to increase Tap-1 by
augmenting the ability of IFN- to activate and increase STAT1
activity and regulate IFN--inducible genes.
Studies from our laboratory have shown that stimulation of the human
macrophage cell line THP-1 with IFN- and LPS resulted in a
synergistic increase in Tap-1 gene transcription, mRNA, and protein
expression. To further understand the mechanism controlling the ability
of LPS to augment the IFN- response, we initiated studies on the
Tap-1 promoter. Our results suggest that induction of Tap-1 gene
expression may be controlled by a unique mechanism in macrophages.
Previous studies on the regulation of the Tap-1 promoter by IFN- in
HeLa cells have been conflicting (4, 9). These two reports
appear to disagree about the importance of STAT1 binding to the GAS
site or of IRF-1 binding to the ISRE site for the regulation of this
promoter by IFN-. Using deletion and mutation analysis of either the
ISRE or the GAS site, the initial report concluded that the GAS site
mediated the IFN- response in HeLa cells (4).
Transfection experiments using mutant promoter plasmids showed the
importance of the GAS site in response to IFN-. Band-shift
experiments showed that STAT1-binding activity was induced after 1
h in this cell line and STAT1 could bind the human Tap-1 promoter GAS
site. They also showed an induction of IRF-1-binding activity after
stimulation of HeLa cells for 16 h. The conclusion of this study,
that STAT1 is important for the response of the human Tap-1 promoter to
IFN- in HeLa cells, is consistent with our finding in the macrophage
line THP-1. However, the second report suggested that IRF-1 binding to
the Tap-1 promoter was more important for the response of this gene to
IFN- (9). In this work, the authors defined the
critical region for induction by IFN- as an IRF-E site, which
encompassed parts of both the ISRE and GAS sites, as we have defined
the region in this study. Transfection results showed that mutation of
the IRF-E site blocked the ability of the promoter to respond to
IFN- after stimulation for 24 h. Using in vivo footprinting of
HeLa cells stimulated for 5 or 18 h, they showed protected G
residues throughout this IRF-E region of the Tap-1 promoter. They also
showed an enhanced band, which by our definition of this region would
be exactly in between the ISRE and GAS sites. Band-shift analysis
showed IRF-1 binding to the IRF-E oligonucleotide after 5 h of
stimulation with IFN-. Their results also show another band higher
up in the gel, which was not discussed. Based on mobility in their
band-shift gels, it could be STAT1. Using IRF-1 knockout mice
splenocytes, they showed a reduced basal level in the expression of
Tap-1 and LMP2 mRNA. They did not show whether Tap-1 expression was
still inducible in any cell type from these mice. Thus, they conclude
that IRF-1 is critical for the regulation of Tap-1 gene expression
following stimulation with IFN-. Surprisingly, although both of
these groups used HeLa cells and similar techniques, they have
different conclusions regarding the regulation of the Tap-1 gene. More
recently, a third report has studied the bidirectional promoter in
three melanoma cell lines that differently express LMP2 and Tap-1
(13). In all three lines, Tap-1 RNA was detected by RT-PCR
regardless of whether the cells had been stimulated with IFN-. In
contrast, LMP2 RNA was constitutively expressed in one line, induced by
stimulation with IFN- in a second line, and it was not expressed
under any conditions tested in the third cell line. Based on their
transfection and band-shift data, they concluded that either STAT1 or
IRF-1 needs to be present for Tap-1 expression, but both factors need
to be present for LMP2 expression.
Unlike many other cell lines, Tap-1 is expressed at almost undetectable
levels in the macrophage cell line THP-1. To present Ags and generate
an immune response, Tap-1 expression needs to be induced. Our studies
on the Tap-1 promoter suggest that STAT1 is important for the initial
induction of transcription for this gene in macrophages. We showed
using band-shift analysis that STAT1-binding activity is rapidly
induced in THP-1 cells following stimulation with IFN-. Mutation of
the GAS site blocks the ability of the promoter to respond to IFN-.
This mutation also prevented most of the synergistic response to
stimulation with IFN- and LPS. However, mutation of the ISRE site
augmented the response to these stimuli. Our data also showed that
STAT1 bound to the combination ISRE/GAS site, further supporting its
role in the initial regulation of the Tap-1 gene in macrophages. STAT1
is known to activate the expression of the IRF-1 gene (14, 15). Based on previous studies in HeLa cells (9),
it is possible that once IRF-1 is produced, it may bind to this region
of the Tap-1 promoter. Once bound, it could potentially play a role in
maintaining an increase in Tap-1 gene expression. Alternatively, IRF-1
may be more important for the induction of Tap-1 gene transcription in
endothelial cells and other nonprofessional APCs.
Our results suggest that LPS can augment an IFN- response in
macrophages. Specifically, this combination of stimuli increased the
transcription of the Tap-1 gene through a GAS site that binds STAT1.
Recent reports from other groups support the idea that IFN- and LPS
may work together to increase the expression of specific genes and
alter cellular responses. However, the proposed mechanisms differ
depending on the gene of interest and the model used. For example, it
has been shown that the combination of LPS and IFN- results in
higher levels of inducible NO synthase (iNOS) induction, as compared
with stimulation with either alone, following stimulation of elicited
peritoneal mouse macrophages and the murine macrophage cell line RAW
264.7 (16, 17). These studies showed that LPS stimulated
the release of IFN-ß from the stimulated cells, which bound to the
cells and further increased STAT1-binding activity and iNOS expression
(16). Additional data have shown that the phosphatase
SHP-1 may regulate the ability of both types of IFNs to activate Tap-1
gene expression, by modulating the STAT1 response (18). In
contrast, studies have shown that normally nontoxic doses of LPS from
specific pathogens can be lethal for mice when given to the mice in
conjunction with IFN- (19, 20). These nontoxic doses
also failed to stimulate iNOS expression in RAW 264.7 cells, as
compared with LPS that was toxic to mice. Using this macrophage cell
line as a model, stimulation with both IFN- and a nontoxic LPS
showed an increase in the activity of STAT1, based on band-shift and
reporter assays using isolated STAT1 binding sites (20).
The mechanism appeared to partially involve the release of either TNF
or IL-1 from the murine macrophage cell line, which further activated
the cells. Finally, in another report, the combination of IFN- and
LPS was shown to increase the levels of expression of the IFN consensus
sequence binding protein gene in peritoneal macrophages
(21). An effect on STAT1 activation was not directly
measured in this study.
From these studies and ours, it is clear that IFN- and LPS can
increase the level of expression of several genes, resulting in the
increased ability of macrophages to respond and participate in both the
innate and acquired immune responses. Based on our data and others, it
appears that STAT1 activation is important for these combined
responses. Future studies will help define how LPS signals combine with
IFN- signals to increase the amount of STAT1 in the nucleus.
Additional experiments will also be helpful in delineating whether this
is solely through the activation of STAT1 directly by the IFN- and
LPS signaling pathways, or by the release of soluble factors whose
signaling pathways work through STAT1.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Microbiology and Immunology, University of North Carolina School of Medicine, Chapel Hill, NC.
3 Address correspondence and reprint requests to Dr. Michael J. Klemsz, Department of Microbiology and Immunology, Indiana University School of Medicine, 635 Barnhill Drive, MS5010, Indianapolis, IN 46202.
4 Abbreviations used in this paper: LMP2, low molecular mass polypeptide 2; GAS, IFN- activation site; iNOS, inducible NO synthase; IRF, IFN regulatory protein; ISRE, IFN-stimulated regulatory element; TK, thymidine kinase.
Received for publication November 5, 1999. Accepted for publication June 23, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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  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]
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M.-C. Rouyez, M. Lestingi, M. Charon, S. Fichelson, A. Buzyn, and I. Dusanter-Fourt IFN Regulatory Factor-2 Cooperates with STAT1 to Regulate Transporter Associated with Antigen Processing-1 Promoter Activity J. Immunol., April 1, 2005; 174(7): 3948 - 3958. [Abstract] [Full Text] [PDF]
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P. W. O'Donnell, A. Haque, M. J. Klemsz, M. H. Kaplan, and J. S. Blum Cutting Edge: Induction of the Antigen-Processing Enzyme IFN-{gamma}-Inducible Lysosomal Thiol Reductase in Melanoma Cells Is STAT1-Dependent but CIITA-Independent J. Immunol., July 15, 2004; 173(2): 731 - 735. [Abstract] [Full Text] [PDF]
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L. Marques, M. Brucet, J. Lloberas, and A. Celada STAT1 Regulates Lipopolysaccharide- and TNF-{alpha}-Dependent Expression of Transporter Associated with Antigen Processing 1 and Low Molecular Mass Polypeptide 2 Genes in Macrophages by Distinct Mechanisms J. Immunol., July 15, 2004; 173(2): 1103 - 1110. [Abstract] [Full Text] [PDF]
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 A. A. Cecil and M. J. Klemsz p38 activation through Toll-like receptors modulates IFN-{gamma}-induced expression of the Tap-1 gene only in macrophages J. Leukoc. Biol., March 1, 2004; 75(3): 560 - 568. [Abstract] [Full Text] [PDF]
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K. Schroder, P. J. Hertzog, T. Ravasi, and D. A. Hume Interferon-{gamma}: an overview of signals, mechanisms and functions J. Leukoc. Biol., February 1, 2004; 75(2): 163 - 189. [Abstract] [Full Text] [PDF]
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