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,
,§
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Department of Microbiology, Immunology, and Molecular Genetics,
Division of Dermatology,
Molecular Biology Institute, and
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Howard Hughes Medical Institute, University of California, Los Angeles, School of Medicine, Los Angeles, CA 90095; and
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Immunobiology Center, Mount Sinai School of Medicine, New York, NY 10029
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(6, 7, 8, 9, 10).
IL-10 has pleiotropic functions in various hemopoetic cell types. While
having positive effects on B cell and CD8+ T cell
function (11, 12), IL-10 is best known for mediating the
down-regulation of Th1 responses by inhibiting the production of
macrophage IL-12, NK-cell IFN-
, and most proinflammatory cytokines
(1, 11, 13, 14, 15, 16, 17, 18, 19). This function is readily apparent in the
IL-10-/- mouse, which acquires autoimmune
manifestations of colitis due to an overabundance of IL-12 and IFN-
(20, 21).
The immunosuppressive functions of IL-10 are an important mechanism for protecting the host from harmful effects of prolonged inflammatory responses in the context of microbial infection (13, 15). Moreover, modulation of IL-10 expression can influence the host’s susceptibility to disease (21). For example, IL-10 is strongly expressed at the site of disease in patients with severe mycobacterial infection (e.g., lepromatous leprosy and tuberculosis) and can down-regulate mycobacterial-specific Th1 responses (5). Furthermore, polymorphisms in the human IL-10 promoter have been identified that correlate IL-10 expression patterns with the severity of multiple autoimmune disorders such as lupus (22, 23, 24), rheumatoid arthritis (25), and various cancers (26, 27, 28, 29). These findings, combined with the results obtained with the IL-10 knockout and IL-10 transgenic mice (12, 30, 31), exemplify how alterations in IL-10 expression can influence immune responses.
One intriguing feature of macrophage biology is the ability of
activated macrophage populations to produce both proinflammatory
cytokines, such as IL-12, TNF-, and IL-1, and antiinflammatory
cytokines, including IL-10 and TGF-ß. The balance of pro- and
antiinflammatory cytokine expression is of central importance for
understanding how the immune system regulates responses to pathogenic
infection. The inducible promoters of several proinflammatory cytokine
genes have been characterized extensively and have been found to be
regulated by a similar set of transcription factor families, including
the Rel, C/EBP,3 and
AP-1 families (32, 33, 34, 35, 36, 37, 38, 39, 40). In contrast, much less is known
about the regulation of antiinflammatory cytokine genes. Interestingly,
experiments performed with chemical inhibitors of Rel proteins or
overexpressed I-
B (inhibitory protein that dissociates from
NF-B) suggest that the Rel family does not contribute to IL-10 gene
induction in macrophages (41, 42).
To gain additional insight into antiinflammatory cytokine gene regulation, we chose to characterize at the molecular level the mechanisms underlying IL-10 gene induction in stimulated macrophages. We present a comprehensive functional characterization of the murine IL-10 promoter in the macrophage-like cell line, RAW264.7. Thorough mutagenesis revealed that a single control element is essential for the induction of the IL-10 promoter in response to LPS. This control element, located between -89 and -78 relative to the transcription start site, interacts with Sp1 family members in RAW264.7 nuclear extracts. Multiple lines of evidence support the functional relevance of Sp1 for IL-10 promoter function and suggest that Sp1 may be a direct mediator of induction. This study provides an initial step toward a detailed analysis of the pathways responsible for IL-10 induction and the differential regulation of pro- and antiinflammatory cytokine genes in macrophages.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A 1.6-kb fragment of the IL-10 promoter (-1538/+64) was amplified by PCR from mouse genomic DNA and subsequently cloned into the XhoI and HindIII sites of the pGL2B polylinker (Promega, Madison, WI). The cloned sequence was found to be identical with that reported previously (43). Promoter deletion mutants were amplified from the -1536/+64 promoter clone by PCR, using an upstream primer containing an XhoII restriction site and a downstream primer containing a HindIII site. The PCR products were subsequently inserted into the luciferase reporter vector pGL2B. Most substitution mutants were generated by a two-step PCR procedure using overlapping internal primers that contain a mutant sequence, as described previously (37, 44). All IL-10 promoter constructs used in chloramphenicol acetyltransferase (CAT) assays were cloned from pGL2B into pCAT basic (Promega), which was modified to contain XhoI and HindIII sites in the polylinker. All PCR-generated inserts were sequenced before their use. All plasmids used in transient transfection assays were purified using an endotoxin-free purification system (Qiagen, Valencia, CA).
The expression plasmids containing the Gal4 DNA binding domain (amino acids 1–147) fused at the N terminus of the VP-16, CTF, and TAT activation domains were described previously (45, 46). All plasmids that express the Gal4 DNA binding domain (amino acids 1–147) fused to various Sp1 domains were reported previously. Gal4-Sp1, Gal4-Sp1A, Gal4-Sp1B, Gal4-Sp1BC, and Gal4-Sp1BCC contain amino acids 83–778, 83–262, 263–542, 425–542, and 484–542, respectively (45, 47, 48).
Cell lines and reagents
The RAW264.7 murine macrophage line (American Type Culture Collection, Manassas, VA) was maintained in DMEM supplemented with 10% FBS (Omega, Tarzana, CA) (assayed for low endotoxin activity), penicillin/streptomycin, and glutamine. LPS (Salmonella typhosa) and cycloheximide (CHX) were from Sigma (St. Louis, MO).
RT-PCR and ELISA
Total RNA was isolated from RAW264.7 cells (Qiagen RNeasy mini kit). IL-10 cDNA was derived from 5 µg of total RNA by reverse transcription using Superscript (Life Technologies, Gaithersburg, MD) and an oligo(dT) primer. PCR was performed on 0.6 µg of cDNA using specific primers, yielding a 500-bp product. The following IL-10-specific primer sequences were used: 5'-CGT CGG ATC CGC CAT GCC TGG CTC ACC ACT GCT-3' and 5'-CGT CTC TAG ATT AGC TTT TCA TTT TGA TCA-3'. PCR was conducted using a standard PCR protocol for 32 cycles. An equal aliquot of cDNA was amplified for 32 cycles using ß-actin primers: 5'-CCT AAG GCC AAC CGT GAA AAG-3' and 5'-TCT TCA TGG TGC TAG GAG CCA-3' (49). The specific ß-actin PCR product was 623 bp. In CHX experiments, RAW264.7 cells were pretreated with CHX (10 µg/ml) for 15 min before stimulation with LPS. Aliquots of PCR products were separated on a 1.2% agarose gel, and visualized after ethidium bromide staining with UV light.
Murine IL-10 protein was measured from 1 x 106 RAW264.7 cells cultured in a six-well plate in 2 ml of DMEM (10% FCS, penicillin/streptomycin, glutamine) and activated with LPS for 24 h. A total of 100 µl was then tested by murine IL-10 ELISA (PharMingen, San Diego, CA).
Transfection
RAW264.7 cells were transiently transfected using the Superfect transfection reagent (Qiagen). For transfection with luciferase reporter plasmids, 2.5 x 106 cells were plated in a six-well plate. The following day, the cells were washed with PBS and transfected with 2 µg of reporter plasmid and 0.5 µg of heat shock promoter ß-galactosidase (ß-gal) reporter (provided by Bradley Cobb, University of California, Los Angeles). DNA was incubated in DMEM medium without serum or antibiotics (100 µl) with Superfect, at a 1:3 ratio (µg of DNA to µl of Superfect), for 5–10 min at room temperature. A total of 600 µl of complete DMEM (10% FBS, penicillin/streptomycin, glutamine) was added to the DNA/Superfect mix, then added dropwise to the cells and incubated at 37°C for 2.5–3 h. Cells were washed with PBS and split into two wells in 2.5 ml of complete DMEM. The cells in one of the two wells was activated with LPS (5 µg/ml) 6 h posttransfection and incubated for 24 h, after which all cells were harvested. For reporter assays, whole cell extracts were prepared using 1x cell reporter lysis buffer (Promega). Luciferase activity was determined from 40 µl of cell extract, and ß-gal activity from 30 µl of extract, as per the Promega protocol. Transient transfection assays with CAT reporter plasmids were performed by the same protocol with the following exceptions: 1) 12 x 106 cells were plated in a 100-mm dish; 2) RAW264.7 cells were transfected with 10 µg of reporter plasmid and 2.5 µg of heat shock promoter ß-gal reporter in incomplete DMEM (300 µl); 3) cells were then incubated with the DNA/superfect mix in 3 ml of complete DMEM; and 4) each transfection was divided into two 100-mm plates in 10 ml complete DMEM (10% FCS/penicillin-streptomycin/glutamine). CAT assays were performed with 100–250 µg of total protein from cell lysates, as per the Promega TLC protocol. Quantitation of the conversion of 14C-chloramphenicol to its acetylated forms was performed by PhosphorImager analysis (Molecular Dynamics).
Nuclear extracts and DNA-binding assays
RAW264.7 nuclear extracts were prepared by a modification of the
method of Dignam et al. (50), as previously described
(37, 51). Extraction and dialysis buffers were
supplemented with 1 mM PMSF, 0.5 µg/ml leupeptin, 1 µg/ml
aprotinin, and 1 µM pepstatin (Sigma). EMSA probes were prepared by
annealing single-stranded oligonucleotides (Life Technologies) that had
been gel purified. Probes (200 ng) were labeled using polynucleotide
kinase and [-32P]ATP. The labeled probes
were purified with a NucTrap purification column (Stratagene, La Jolla,
CA). Sequences of IL-10 wild-type and mutant EMSA probes are displayed
in Fig. 5
A. For Sp1/Sp3 EMSAs, probe (5 x
105 cpm) was added to 5 µg of nuclear extract
with 1 µg poly(dI.dC), in binding buffer (10 mM Tris, pH 7.5, 50 mM
NaCl, 1 mM DTT, 1 mM EDTA, 5% glycerol). Binding was performed at room
temperature for 30 min. Protein-DNA complexes were separated on a 4%
polyacrylamide, 0.1x TBE gel run at room temperature for 1.5 h at
150 V. Unlabeled oligonucleotide competitors were added to nuclear
extracts, poly(dI.dC), and binding buffer at 100- to 200-fold molar
excess for 30 min before addition of labeled probe. For supershift
experiments, 4 µg of anti-Sp1 mAb or 2 µg of anti-Sp3
antisera, 1 µg poly(dI.dC), and binding buffer were added to nuclear
extracts for 30 min at room temperature before addition of labeled
probe. mAbs were obtained from Santa Cruz Biotechnologies (Santa Cruz,
CA). Purified human rSp1 was obtained from Promega.
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RAW264.7 cells were either left unactivated or stimulated with
LPS. After 18 h, the cells were incubated with Brefeldin A (10
µg/ml) (Sigma) for 6 h. After a total of 24 h, the cells
were harvested, washed twice with cold PBS, and fixed in 50 µl
PBS-2% FCS and 100 µl 4% formaldehyde fixing solution for 20 min at
room temperature. After washing with PBS, cells were incubated in 300
µl permeabilization buffer (PB) (1x PBS, 10% FCS, 10% saponin) for
30 min at room temperature. Cells were then centrifuged, washed in PB
buffer, and centrifuged once again. Cells were resuspended in 50 µl
of PB buffer with 1 µl of FITC rat anti-mouse IL-10 (
0.5 µg
Ab, rat IgG2a) or IgG2a control Ab. The mixture was incubated for 30
min at room temperature. The cells were washed in PB buffer and then
twice in PBS-10% FCS. Cells were finally resuspended in 100 ml of
PBS-2% FCS + 100 µl of 2% paraformaldehyde and analyzed by flow
cytometry.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies demonstrated that IL-10 mRNA and protein are
induced in mouse and human primary macrophages and cell lines in
response to LPS (13, 52). To study the transcriptional
regulatory mechanisms for the murine IL-10 gene, the murine
macrophage-like cell line RAW264.7 was selected because it was
previously found to express IL-10 (52) and has been used
successfully for transfection studies of other promoters. The induction
of IL-10 expression was confirmed by monitoring mRNA and protein levels
following stimulation with LPS. IL-10 mRNA levels were determined by
RT-PCR over a time course of activation from 0–24 h. The PCR
product was undetectable in unactivated cells (Fig. 1A, lane 1), but
was observed 2 h following LPS stimulation (Fig. 1A,
lane 2). All cDNA preparations were normalized to ß-actin
mRNA levels (Fig. 1A). IL-10 protein, as measured by ELISA,
was observed 4 h following LPS stimulation (Fig. 1B). The protein concentrations were comparable with those
obtained with primary macrophages derived from PBMC (9),
suggesting that RAW264.7 cells can serve as an appropriate model system
for studying IL-10 regulation.
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C, lanes 4 and
5), suggesting that IL-10 transcription depends on the
induced synthesis of at least one protein. Alternatively, IL-10
transcription may depend on a constitutively expressed, labile protein
that rapidly degrades following addition of CHX. Identification of DNA sequences required for the induction of IL-10 promoter activity
Most inducible cytokine genes appear to contain DNA elements
within their promoters that are of central importance for gene
induction (37, 38, 39, 40, 53, 54). To determine whether the IL-10
promoter can mediate transcriptional induction in LPS-activated
RAW264.7 cells, a transient transfection assay was employed. A promoter
fragment extending from nucleotide -1538 to nucleotide +64, relative
to the +1 transcription start site (43), was inserted into
a CAT reporter vector (pCAT; Promega). Following transfection of
RAW264.7 cells and activation with LPS for 24 h, CAT activity was
measured. The results revealed an 8-fold increase in CAT activity upon
LPS treatment (Fig. 2A), which
was 15–20-fold greater than the activity of the promoterless
vector (pCAT). These results are consistent with the hypothesis that
the promoter contributes to the induction of IL-10 transcription. As a
control, the activity of the CMV promoter did not increase
significantly (Fig. 2A).
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B. Following transfection of RAW264.7
cells with the luciferase reporter plasmid containing IL-10 promoter
sequence from -1538 to +64, luciferase activity was detected following
LPS activation that was 50–100-fold greater than the activity detected
with the promoterless vector (Fig. 2B, compare -1538/+64
with pGL2B). The activity of this IL-10 promoter fragment was
20–50-fold higher in LPS-stimulated cells than in unactivated cells
(data not shown).
Analysis of the 5' deletion mutants revealed that deletion of sequences
between -1538 and -118 reduced promoter activity by less than 2-fold
(Fig. 2B, compare -1538/+64 with -118/+64). In contrast,
promoter activity was reduced to background levels by deletion of
sequences between -118 and -78. From the 3' end, sequences between
+64 and +9 were deleted with little effect on promoter activity (Fig. 2B). These results reveal that the DNA sequences between
-118 and +9 are sufficient for strong promoter activity in
LPS-activated RAW264.7 cells, and that at least one critical DNA
element is present between -118 and -78.
A comparison of luciferase activities in unactivated and LPS-activated
macrophages revealed strong induction of the -118/+64 mutant (data not
shown). This finding suggests that DNA elements that are critical for
promoter induction are located downstream of -118. However, this
result is inconclusive because of an induction artifact that has been
observed with all luciferase reporter genes in RAW264.7 cells
(37). To confirm that the sequences downstream of -118
are indeed sufficient for IL-10 promoter induction, the -118/+64
deletion mutant was therefore inserted into the CAT reporter vector.
The transfection results revealed an 8-fold induction of this mutant
promoter by LPS, comparable with the induction observed with the
-1538/+64 promoter (Fig. 2A). Furthermore, the strength of
the -118/+64 mutant in LPS-activated cells was comparable with that of
the -1538/+64 promoter (Fig. 2A, compare -118/+64 with
-1538/+64).
Localization of the DNA sequences required for LPS-induced promoter activity by substitution mutant analysis
To localize DNA elements downstream of -118 that are required for
IL-10 promoter activity, a series of 6–10-bp substitution mutants
scanning the region between -118 and the TATA box at -32 was prepared
(Fig. 3A). One mutation,
-11/-6, was introduced downstream of the TATA box. All substitution
mutants were prepared in the context of the -118/+64 promoter fragment
and were placed upstream of the luciferase reporter gene (before the
luciferase artifact was discovered (37)). Following
transient transfection of RAW264.7 cells and activation with LPS, three
mutants (-98/-89, -88/-79, -78/-69) exhibited significantly
reduced promoter activities (5–20% of wild type; Fig. 3B).
The activities of the other mutants were within 2-fold of the wild-type
promoter activity.
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may include a
recognition site for one transcription factor or for multiple factors.
To gain insight into the number of recognition sites within this
region, a series of 3- and 4-bp substitution mutants between -102 and
-68 was prepared in the context of a luciferase reporter (Fig. 4A). Strongly reduced promoter
activities were observed with four mutants, which contain altered
sequences between -89 and -78 (Fig. 4B, mutants -89/-86,
-86/-84, -83/-81, and -80/-78). These results suggest that the
critical element spans a minimum of 7 bp (-86 to -80) and a maximum
of 12 bp (-89 to -78), most consistent with the presence of
recognition sites for one or two proteins.
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C) reveal minimal induction of mutants that disrupt the
defined element (-89/-86, -86/-84, -83/-81, and -80/-78) and
strong induction of mutants that alter flanking sequences (-92/-90
and -77/-75). These results are consistent with a critical role for
the element between -89 and -78 during IL-10 promoter induction. Correlation between DNA-binding activities and IL-10 promoter activity
EMSAs with RAW264.7 nuclear extracts were used to identify
potential regulators of the -89/-78 control element. Furthermore, as
one initial test of the functional relevance of the EMSA complexes
identified, the mutant series described above was analyzed; a simple
prediction is that the nucleotides recognized by the functionally
relevant DNA-binding protein should correspond to the nucleotides
required for promoter activity in the transient transfection assay.
Radiolabeled probes extending from -106 to -59, containing wild-type
and mutant DNA sequences, were prepared and used for EMSAs (Fig. 5A). The wild-type probe
yielded multiple protein-DNA complexes in nuclear extracts from
LPS-stimulated RAW264.7 cells (Fig. 5B, lane 1).
However, only two complexes, including one abundant complex (complex 1)
and one complex that was barely detectable (complex 2), were reduced by
the same mutations that eliminated promoter function (compare Fig. 5B, lanes 2–9, with Fig. 4, B and
C). The sensitivity of complex 2 to the key mutations is not
easily apparent in the experiment shown, but was revealed by the
consistent absence of this complex in multiple experiments performed
with the mutant probes (data not shown). The proteins within complexes
1 and 2 are therefore viable candidates for the relevant activator(s)
of IL-10 transcription.
Interestingly, a database analysis with the important 12-bp sequence
revealed similarity to a nonconsensus recognition site for the Sp1
protein. Although Sp1 is expressed ubiquitously, it has been suggested
to contribute to the induction of several genes, including the IL-1ß
(55), p21CIP1/WAF1,
p15INK4B (56, 57), 2 (I) collagen
(58, 59), and TNFR-II (60) genes. To confirm
that Sp1 can bind the IL-10 promoter element and to determine whether
Sp1 binding requires the nucleotides that are necessary for promoter
function, purified human rSp1 was analyzed by EMSA using the wild-type
and mutant probes. Strikingly, the complex formed with rSp1 required
the same nucleotides for binding as complex 1 (Fig. 5, compare
C with B) and migrated with a similar mobility
(data not shown).
To test the hypothesis that complexes 1 and 2 observed with RAW264.7
nuclear extracts contain Sp1 family members, competition experiments
were conducted with unlabeled oligonucleotides (>100-fold molar
excess) containing the wild-type and mutant IL-10 promoter sequences
(-106/-59), as well as wild-type and mutant Sp1 consensus sequences
from the SV40 enhancer (61). The wild-type IL-10
competitor strongly reduced complex 1, whereas the mutant competitor
(the -83/-81 mutant) only slightly reduced this complex (Fig. 5D, lanes 1–3). Furthermore, the wild-type Sp1
consensus oligonucleotide reduced the complex in a titratable manner,
whereas the mutant consensus oligonucleotide was a much less effective
competitor (Fig. 5D, lanes 4–7). Complex 2
appeared to be affected similarly to complex 1, but was difficult to
detect in these experiments. These results support the hypothesis that
complex 1, and perhaps complex 2, contains Sp1 family members.
To identify the Sp1 family members within complexes 1 and 2, Abs were
added to the binding assays. An Sp1 mAb selectively supershifted
complex 1 (Fig. 5E, lane 2) and an Sp3 Ab
selectively inhibited formation of complex 2 (Fig. 5E,
lane 3). Similar relative mobilities of Sp1 and Sp3 EMSA
complexes were reported in previous studies (57, 58, 59).
Thus, these results implicate Sp1 and Sp3 as potential activators of
the IL-10 promoter through the -89/-78 element.
A consensus Sp1 site can substitute for the critical IL-10 promoter element
To test the hypothesis that Sp1 or Sp3 is a relevant activator of
the IL-10 promoter through the -89/-78 element, the native
nonconsensus Sp1 recognition sequence was replaced with the Sp1
consensus sequence (61, 62), CGGGGCGGGGCG, in the context
of the -1538/+64 and -118/+64 CAT reporter plasmids (Fig. 6A). Following transfection of
RAW264.7 cells and stimulation with LPS, the altered promoters
(-1538/+64Sp1C and -118/+64Sp1C) retained the strong induction
observed with the wild-type IL-10 promoter (-1538/+64WT and
-118/+64WT) (Fig. 6B). This result demonstrates that a
protein that binds the Sp1 consensus sequence can support the inducible
activity of the IL-10 promoter.
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The above results provide evidence that Sp1 is responsible for the
activity of the -89/-78 element within the IL-10 promoter. However,
the only evidence presented to date that Sp1 may be the target of an
LPS-induced signal transduction pathway, and therefore contribute
directly to IL-10 promoter induction, is the exclusive requirement for
the Sp1 site for inducible promoter activity. In other words, the
systematic mutant analysis revealed that the -89/-78 element was the
only essential element for promoter activity and induction, suggesting
that this element may be a direct contributor to induction. To test
this hypothesis, an IL-10 promoter fragment encompassing the Sp1 site
(-118/-58) was placed upstream of a heterologous core promoter
comprised of consensus TATA and initiator (Inr) elements
(63). Transient transfection experiments revealed that
this 60-bp sequence can confer LPS responsiveness to the heterologous
core promoter (-118/-58wtTI), with the LPS responsiveness abolished
by mutation of 3 bp from -83 to -81 (-118/-58 mTI) (Fig. 7A).
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A). The reduced induction could result from core promoter
differences that influence the extent of induction in the transient
assay (e.g., by altering a rate-limiting step). Alternatively, other
promoter elements that were not included in the fragment transferred to
the heterologous promoter may contribute to induction; it is possible
that these other elements were not identified in the systematic mutant
analysis because of functional redundancies with other elements.
Interestingly, strong induction could be restored in the heterologous
context by inserting a dimer of the IL-10-89/-78
element (-89/-78[x2]wtTI) or the Sp1 consensus sequence
(-89/-78[x2]Sp1C TI) (Fig. 7B). This result demonstrates
that the Sp1 element can confer LPS responsiveness, although a dimer
appears to be necessary for strong induction in the heterologous
context. LPS stimulation does not enhance the Sp1 or Sp3 DNA-binding activities
Transcription factors are often induced at the level of their
DNA-binding activities. To determine whether LPS stimulation
results in increased DNA binding of Sp1 or Sp3 to the IL-10
element, EMSAs were performed with nuclear extracts from unactivated
and LPS-stimulated RAW264.7 cells. The Sp1 and Sp3 DNA-binding
activities were not altered in the extracts from LPS-stimulated cells
(4 h) (Fig. 8A). Similar
results were obtained when the time of stimulation was varied from 2 to
9 h (data not shown). These results are consistent with studies of Sp1
during induction of the TNFR-II promoter by LPS in RAW264.7 cells
(60) and of the 2(I) collagen promoter by oncostatin M
in human dermal fibroblasts (59). However, our results
contrast with a reported 2–3-fold increase in Sp1 DNA binding to an
Sp1 consensus element in RAW264.7 cells upon LPS stimulation
(64). Intracellular flow cytometry of unactivated and
LPS-stimulated RAW264.7 cells demonstrated that a high percentage of
the cells within the population were activated to produce IL-10 (Fig. 8B, panel 2). In addition, NF-B- and
C/EBP-binding activities were found to be significantly enhanced in the
same extracts that were used to monitor Sp1 binding (37
and data not shown). Therefore, induction of the Sp1 DNA-binding
activity by LPS was unlikely to have been obscured by a low efficiency
of cell activation.
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Taken together, the above results support a model in which Sp1’s
capacity for transcriptional activation, but not its DNA-binding
activity, is enhanced upon LPS stimulation. To determine whether Sp1
can mediate transcriptional induction in the absence of its DNA binding
domain, RAW264.7 cells were cotransfected with expression plasmids for
a panel of Gal4 DNA binding domain fusion proteins (Fig. 9A) and a reporter plasmid
under the control of five Gal4 binding sites (Gal4[x5]-pCAT).
Reporter activity was monitored in unactivated and LPS-activated
cells.
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B).
Interestingly, this is comparable with the induction observed with the
wild-type IL-10 promoter in the same experiments (Fig. 9B,
-1538/+64). Gal4 fusion proteins containing the VP-16, CTF, and Tat
activation domains were induced by only about 2-fold (Fig. 9B). These results are consistent with the hypothesis that
Sp1 is a direct mediator of induction and that Sp1-mediated induction
does not rely on an increase of DNA-binding activity of Sp1.
To localize the domains of the that contribute to its inducible
activity, Gal4 fusion proteins containing Sp1 fragments were analyzed.
A fusion protein containing the Sp1 B domain was induced by 10-fold
(Fig. 9C), comparable with the induction observed with the
full-length protein. In contrast, the 2-fold induction observed with
the Sp1 A domain (Fig. 9C) was comparable with that observed
with the VP-16, CTF, and Tat domains.
The Sp1 B domain is a well-characterized transcriptional activation
domain containing a glutamine-rich C-terminal region and a
serine-threonine-rich N-terminal region (65). A Gal4
fusion protein containing the N-terminal half of the B domain
(Gal4-Sp1BN) was inactive (Fig. 9C), as previously reported
(48). In contrast, a fusion protein containing the
C-terminal half of the B domain (Gal4-Sp1BC, amino acids 425–542)
supported transcriptional induction by LPS, but the magnitude of the
induction was only about 3-fold. Furthermore, the absolute, induced
activity of this fusion protein was 3-fold less than that of the
Gal4-Sp1B protein (Fig. 9C). The BCC domain exhibited little
activity and no significant induction. These results suggest that the B
domain is the primary mediator of Sp1 induction, and that amino acids
in both the N-terminal and C-terminal halves of this domain are
required for efficient induction.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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One unexpected finding was that only one element upstream of the IL-10 core promoter appears to be essential for promoter activity. Because this finding is contrary to well-established models of combinatorial gene regulation, we strongly suspect that other important elements exist within the promoter that were not revealed by the transient transfection assay. The inhibition of IL-10 induction by CHX supports the hypothesis that additional inducible proteins are required, as the putative posttranslational modification involved in Sp1 induction is unlikely to depend on de novo protein synthesis. Some proteins that are critical for activity of the endogenous IL-10 promoter may not be necessary in the transient assay because of the high plasmid copy number or the aberrant chromatin structure that assembles on transiently transfected plasmids (66). Other important proteins may be redundant with one another. In other words, disruption of one element may have little effect on promoter activity because a nearby, redundant element may remain functional. To address the former possibility, the systematic mutant analysis will need to be repeated using a different assay, such as a stable transfection assay. To address the latter possibility, a different mutant analysis strategy aimed at identifying the elements that are sufficient for inducible activity will need to be employed. It also may be noteworthy that the current analysis does not exclude the possibility that enhanced mRNA stability contributes to IL-10 induction.
Although several DNA elements are likely to contribute to the function of the endogenous IL-10 promoter, the Sp1 element identified in this study is likely to remain among the most important. The data presented suggest that Sp1 may actually be a direct target of a signal transduction pathway that contributes to IL-10 induction. This hypothesis is supported by the exclusive requirement for Sp1 during promoter induction in the transient assay, by the ability of the Sp1 element to confer inducibility to a heterologous promoter, and by the ability of the Sp1 B domain to confer inducibility when fused to a Gal4 DNA binding domain. Similar lines of evidence have been used in previous studies of a few other genes to suggest that Sp1 is a target of signaling pathways involved in inducible transcription (55, 56, 57, 58, 59, 60, 67 ; see Results). In one of these studies, of the p21 and p15 promoters, the inducible function was localized to the B transactivation domain, similar to the results of this analysis (56, 57).
Although the IL-10 promoter analysis and the previous studies cited above provide a significant body of evidence suggesting the Sp1 is a direct contributor to inducible transcription, its precise role remains uncertain. To conclusively establish that Sp1 is a direct target, it will be necessary to identify an inducible posttranslational modification and to then show that disruption of this modification has appropriate functional consequences. This goal may be difficult to achieve because the Sp1 transactivation domains are highly phosphorylated, particularly within the serine-threonine-rich domains (65, 68). Furthermore, Sp1 possesses other types of posttranslational modifications, which may contribute to its inducible activity (69, 70). Finally, it is important to note that the putative signal transduction pathway does not need to modify Sp1 itself. An alternative possibility is that an essential coactivator for Sp1 acquires a posttranslational modification during cell activation, allowing Sp1 to stimulate transcription more effectively. The existence of several Sp1 coactivators have been reported, including CRSP, Rb, and hTAFII130 (47, 71, 72, 73, 74, 75), greatly increasing the challenge of elucidating the mechanism by which Sp1 contributes to transcriptional induction.
Our data support the hypothesis that the molecular regulation of
antiinflammatory cytokine genes is fundamentally different from the
regulation of proinflammatory cytokine genes. This hypothesis was
originally based on the observation that the induction kinetics for the
IL-10 gene and proinflammatory cytokine genes can differ (15, 76, 77). Certain inducers, such as IFN-, have also been found to
affect expression of the IL-10 gene and proinflammatory cytokine genes
differentially (9). Furthermore, indirect evidence has
been obtained, from chemical inhibitor experiments and I-B
overexpression experiments, that NF-B, a key regulator of most
proinflammatory cytokine genes, is not necessary for IL-10 induction
(41, 42). Although considerable variability exists between
the proinflammatory cytokine gene promoters, and although only a subset
has been subjected to comprehensive mutant analyses, clear similarities
have emerged. In particular, most of these promoters contain
functionally important binding sites for Rel, C/EBP, and AP-1 proteins
when analyzed in transient transfection assays (33, 34, 35, 36, 37, 38, 39, 40).
In contrast, using the same type of assay, functionally important
binding sites for these proteins were not found in the IL-10 promoter.
Rather, our study provides evidence that Sp1 may be a key regulator of
IL-10 transcription. These results provide a mechanism that may help
explain the differential production and regulation of pro- and
antiinflammatory cytokines. On the basis of this knowledge, it
eventually may be possible to intervene in a variety of human
diseases.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Stephen T. Smale, Howard Hughes Medical Institute, 6-730 MRL, 675 Charles E. Young Drive South, University of California, Los Angeles, Los Angeles, CA 90095-1662. E-mail address:
3 Abbreviations used in this paper: C/EBP, CCAAT/enhancer-binding protein; ß-gal, ß-galactosidase; CAT, chloramphenicol acetyltransferase; CHX, cycloheximide; Inr, initiator; PB, permeabilization buffer.
Received for publication October 8, 1999. Accepted for publication December 9, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and interleukin-10 by human monocytes stimulated with killed and live Haemophilus influenzae or Streptococcus pneumoniae organisms. Infect. Immun. 67:3714. differentially regulates interleukin-12 and interleukin-10 production in leprosy. J. Clin. Invest. 99:336.[Medline]
. J. Immunol. 157:12.[Abstract]
regulation of experimental Trypanosoma cruzi infection. J. Exp. Med. 175:169.-production by suppressing natural killer cell stimulatory factor/IL-12 synthesis in accessory cells. J. Exp. Med. 178:1041. are costimulators of interferon production by natural killer cells in severe combined immunodeficiency mice with listeriosis, and interleukin-10 is a physiologic antagonist. Proc. Natl. Acad. Sci. USA 90:3725.B in cytokine gene regulation. Adv. Immunol. 65:1.[Medline]
B half-site. Mol. Cell. Biol. 15:5258.[Abstract]
B and c-Ets family members and transcriptional regulation of the cell-specific and inducible macrophage inflammatory protein 1 immediate early gene. Mol. Cell. Biol. 13:5276.B synergistically activate transcription of the inflammatory cytokines, interleukin 6 and interleukin 8. Proc. Natl. Acad. Sci. USA 90:10193.B. Mol. Cell. Biol. 13:7191.B into human macrophages: lipopolysaccharide-induced, but not zymosan-induced, proinflammatory cytokines are inhibited, but IL-10 is nuclear factor-B independent. J. Immunol. 162:2939., MIP-1, IL-12 and nitric oxide production and protects from the lethal effects of endotoxin. Shock 10:49.[Medline]
and nitric oxide production in RAW264.7 macrophages and in endotoxemic mice. J. Immunol. 157:4634.[Abstract]
2(I) collagen gene. Nucleic Acids Res. 25:3712. 2(I) collagen gene via the Sp1/Sp3 binding site. J. Biol. Chem. 272:24666. 2(I) collagen gene expression through a cis-acting element that contains a Sp1-binding site. J. Biol. Chem. 269:14828.This article has been cited by other articles:
|
|
 |
|
  A.-N. N. Ahyi, H.-C. Chang, A. L. Dent, S. L. Nutt, and M. H. Kaplan IFN Regulatory Factor 4 Regulates the Expression of a Subset of Th2 Cytokines J. Immunol., August 1, 2009; 183(3): 1598 - 1606. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Alvarez, C. Municio, S. Alonso, M. Sanchez Crespo, and N. Fernandez The Induction of IL-10 by Zymosan in Dendritic Cells Depends on CREB Activation by the Coactivators CREB-Binding Protein and TORC2 and Autocrine PGE2 J. Immunol., July 15, 2009; 183(2): 1471 - 1479. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. B. Pruett, B. Cheng, R. Fan, W. Tan, and T. Sebastian Oxidative Stress and Sodium Methyldithiocarbamate-Induced Modulation of the Macrophage Response to Lipopolysaccharide In Vivo Toxicol. Sci., June 1, 2009; 109(2): 237 - 246. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Xu, Y. Yang, G. Qiu, G. Lal, Z. Wu, D. E. Levy, J. C. Ochando, J. S. Bromberg, and Y. Ding c-Maf Regulates IL-10 Expression during Th17 Polarization J. Immunol., May 15, 2009; 182(10): 6226 - 6236. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Chakrabarti, A. J. Sadler, N. Kar, H. A. Young, R. H. Silverman, and B. R. G. Williams Protein Kinase R-dependent Regulation of Interleukin-10 in Response to Double-stranded RNA J. Biol. Chem., September 12, 2008; 283(37): 25132 - 25139. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P.-h. Park, H. Huang, M. R. McMullen, K. Bryan, and L. E. Nagy Activation of cyclic-AMP response element binding protein contributes to adiponectin-stimulated interleukin-10 expression in raw 264.7 macrophages J. Leukoc. Biol., May 1, 2008; 83(5): 1258 - 1266. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Csoka, Z. H. Nemeth, L. Virag, P. Gergely, S. J. Leibovich, P. Pacher, C.-X. Sun, M. R. Blackburn, E. S. Vizi, E. A. Deitch, et al. A2A adenosine receptors and C/EBP{beta} are crucially required for IL-10 production by macrophages exposed to Escherichia coli Blood, October 1, 2007; 110(7): 2685 - 2695. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Dong, C. Ivascu, H.-D. Chang, P. Wu, R. Angeli, L. Maggi, F. Eckhardt, L. Tykocinski, C. Haefliger, B. Mowes, et al. IL-10 Is Excluded from the Functional Cytokine Memory of Human CD4+ Memory T Lymphocytes J. Immunol., August 15, 2007; 179(4): 2389 - 2396. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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|
|
|
|
|
|
K. J. Staples, T. Smallie, L. M. Williams, A. Foey, B. Burke, B. M. J. Foxwell, and L. Ziegler-Heitbrock IL-10 Induces IL-10 in Primary Human Monocyte-Derived Macrophages via the Transcription Factor Stat3 J. Immunol., April 15, 2007; 178(8): 4779 - 4785. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Zhou, A. A. Nazarian, J. Xu, D. Tantin, L. M. Corcoran, and S. T. Smale An Inducible Enhancer Required for Il12b Promoter Activity in an Insulated Chromatin Environment Mol. Cell. Biol., April 1, 2007; 27(7): 2698 - 2712. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Asai, Y. Makimura, and T. Ogawa Toll-like receptor 2-mediated dendritic cell activation by a Porphyromonas gingivalis synthetic lipopeptide J. Med. Microbiol., April 1, 2007; 56(4): 459 - 465. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. N. Kremer, A. Kumar, and K. E. Hedin Haplotype-Independent Costimulation of IL-10 Secretion by SDF-1/CXCL12 Proceeds via AP-1 Binding to the Human IL-10 Promoter J. Immunol., February 1, 2007; 178(3): 1581 - 1588. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Gee, J. B. Angel, S. Mishra, M. A. Blahoianu, and A. Kumar IL-10 Regulation by HIV-Tat in Primary Human Monocytic Cells: Involvement of Calmodulin/Calmodulin-Dependent Protein Kinase-Activated p38 MAPK and Sp-1 and CREB-1 Transcription Factors J. Immunol., January 15, 2007; 178(2): 798 - 807. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Yang, D. M. Mosser, and X. Zhang Activation of the MAPK, ERK, following Leishmania amazonensis Infection of Macrophages J. Immunol., January 15, 2007; 178(2): 1077 - 1085. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Gee, J. B. Angel, W. Ma, S. Mishra, N. Gajanayaka, K. Parato, and A. Kumar Intracellular HIV-Tat Expression Induces IL-10 Synthesis by the CREB-1 Transcription Factor through Ser133 Phosphorylation and Its Regulation by the ERK1/2 MAPK in Human Monocytic Cells J. Biol. Chem., October 20, 2006; 281(42): 31647 - 31658. [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]
|
|
|
|
|
|
X. Zhang, J. P. Edwards, and D. M. Mosser Dynamic and Transient Remodeling of the Macrophage IL-10 Promoter during Transcription J. Immunol., July 15, 2006; 177(2): 1282 - 1288. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Shoemaker, M. Saraiva, and A. O'Garra GATA-3 Directly Remodels the IL-10 Locus Independently of IL-4 in CD4+ T Cells J. Immunol., March 15, 2006; 176(6): 3470 - 3479. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. W. Lee, S. Bennouna, and E. Y. Denkers Screening for Toxoplasma gondii-Regulated Transcriptional Responses in Lipopolysaccharide-Activated Macrophages Infect. Immun., March 1, 2006; 74(3): 1916 - 1923. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. K. Pathak, S. Basu, A. Bhattacharyya, S. Pathak, M. Kundu, and J. Basu Mycobacterium tuberculosis Lipoarabinomannan-mediated IRAK-M Induction Negatively Regulates Toll-like Receptor-dependent Interleukin-12 p40 Production in Macrophages J. Biol. Chem., December 30, 2005; 280(52): 42794 - 42800. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. C. Taggart, S.-A. Cryan, S. Weldon, A. Gibbons, C. M. Greene, E. Kelly, T. B. Low, S. J. O'Neill, and N. G. McElvaney Secretory leucoprotease inhibitor binds to NF-{kappa}B binding sites in monocytes and inhibits p65 binding J. Exp. Med., December 19, 2005; 202(12): 1659 - 1668. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. H. Nemeth, C. S. Lutz, B. Csoka, E. A. Deitch, S. J. Leibovich, W. C. Gause, M. Tone, P. Pacher, E. S. Vizi, and G. Hasko Adenosine Augments IL-10 Production by Macrophages through an A2B Receptor-Mediated Posttranscriptional Mechanism J. Immunol., December 15, 2005; 175(12): 8260 - 8270. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. A. Jones and R. A. Flavell Distal Enhancer Elements Transcribe Intergenic RNA in the IL-10 Family Gene Cluster J. Immunol., December 1, 2005; 175(11): 7437 - 7446. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Riemann, R. Endres, S. Liptay, K. Pfeffer, and R. M. Schmid The I{kappa}B Protein Bcl-3 Negatively Regulates Transcription of the IL-10 Gene in Macrophages J. Immunol., September 15, 2005; 175(6): 3560 - 3568. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Goffin, D. Demonte, C. Vanhulle, S. de Walque, Y. de Launoit, A. Burny, Y. Collette, and C. Van Lint Transcription factor binding sites in the pol gene intragenic regulatory region of HIV-1 are important for virus infectivity Nucleic Acids Res., August 1, 2005; 33(13): 4285 - 4310. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Saraiva{paragraph}, J. R. Christensen, A. V. Tsytsykova, A. E. Goldfeld, S. C. Ley, D. Kioussis, and A. O'Garra Identification of a Macrophage-Specific Chromatin Signature in the IL-10 Locus J. Immunol., July 15, 2005; 175(2): 1041 - 1046. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Zhang, M. Martin, S. M. Michalek, and J. Katz Role of Mitogen-Activated Protein Kinases and NF-{kappa}B in the Regulation of Proinflammatory and Anti-Inflammatory Cytokines by Porphyromonas gingivalis Hemagglutinin B Infect. Immun., July 1, 2005; 73(7): 3990 - 3998. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Wilmanski, M. Siddiqi, E. A. Deitch, and Z. Spolarics Augmented IL-10 production and redox-dependent signaling pathways in glucose-6-phosphate dehydrogenase-deficient mouse peritoneal macrophages J. Leukoc. Biol., July 1, 2005; 78(1): 85 - 94. [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]
|
|
|
|
 |
|
 X. Wang, M. D. Garrick, F. Yang, L. A. Dailey, C. A. Piantadosi, and A. J. Ghio TNF, IFN-{gamma}, and endotoxin increase expression of DMT1 in bronchial epithelial cells Am J Physiol Lung Cell Mol Physiol, July 1, 2005; 289(1): L24 - L33. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. L. Chelvarajan, S. M. Collins, J. M. Van Willigen, and S. Bondada The unresponsiveness of aged mice to polysaccharide antigens is a result of a defect in macrophage function J. Leukoc. Biol., April 1, 2005; 77(4): 503 - 512. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Cao, J. Liu, L. Song, and X. Ma The Protooncogene c-Maf Is an Essential Transcription Factor for IL-10 Gene Expression in Macrophages J. Immunol., March 15, 2005; 174(6): 3484 - 3492. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z.-Y. Wang, H. Sato, S. Kusam, S. Sehra, L. M. Toney, and A. L. Dent Regulation of IL-10 Gene Expression in Th2 Cells by Jun Proteins J. Immunol., February 15, 2005; 174(4): 2098 - 2105. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. S. K. Yee, Y. Yao, Q. Xu, B. McCarthy, D. Sun-Lin, M. Tone, H. Waldmann, and C.-H. Chang Enhanced Production of IL-10 by Dendritic Cells Deficient in CIITA J. Immunol., February 1, 2005; 174(3): 1222 - 1229. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Mostecki, B. M. Showalter, and P. B. Rothman Early Growth Response-1 Regulates Lipopolysaccharide-induced Suppressor of Cytokine Signaling-1 Transcription J. Biol. Chem., January 28, 2005; 280(4): 2596 - 2605. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Wessells, M. Baer, H. A. Young, E. Claudio, K. Brown, U. Siebenlist, and P. F. Johnson BCL-3 and NF-{kappa}B p50 Attenuate Lipopolysaccharide-induced Inflammatory Responses in Macrophages J. Biol. Chem., November 26, 2004; 279(48): 49995 - 50003. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. W. Steinke, E. Barekzi, J. Hagman, and L. Borish Functional Analysis of -571 IL-10 Promoter Polymorphism Reveals a Repressor Element Controlled by Sp1 J. Immunol., September 1, 2004; 173(5): 3215 - 3222. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Wu, G. A. Zimmerman, S. M. Prescott, and D. M. Stafforini The p38 MAPK Pathway Mediates Transcriptional Activation of the Plasma Platelet-activating Factor Acetylhydrolase Gene in Macrophages Stimulated with Lipopolysaccharide J. Biol. Chem., August 20, 2004; 279(34): 36158 - 36165. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Ohkusu-Tsukada, N. Tominaga, H. Udono, and K. Yui Regulation of the Maintenance of Peripheral T-Cell Anergy by TAB1-Mediated p38{alpha} Activation Mol. Cell. Biol., August 15, 2004; 24(16): 6957 - 6966. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Rothwell, J. R. Young, R. Zoorob, C. A. Whittaker, P. Hesketh, A. Archer, A. L. Smith, and P. Kaiser Cloning and Characterization of Chicken IL-10 and Its Role in the Immune Response to Eimeria maxima J. Immunol., August 15, 2004; 173(4): 2675 - 2682. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. B. Pruett, C. Schwab, Q. Zheng, and R. Fan Suppression of Innate Immunity by Acute Ethanol Administration: A Global Perspective and a New Mechanism Beginning with Inhibition of Signaling through TLR3 J. Immunol., August 15, 2004; 173(4): 2715 - 2724. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. L. Chelvarajan, S. M. Collins, I. E. Doubinskaia, S. Goes, J. Van Willigen, D. Flanagan, W. J. S. de Villiers, J. S. Bryson, and S. Bondada Defective macrophage function in neonates and its impact on unresponsiveness of neonates to polysaccharide antigens J. Leukoc. Biol., June 1, 2004; 75(6): 982 - 994. [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]
|
|
|
|
|
|
M. N. Bradley, L. Zhou, and S. T. Smale C/EBP{beta} Regulation in Lipopolysaccharide-Stimulated Macrophages Mol. Cell. Biol., July 15, 2003; 23(14): 4841 - 4858. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y.-W. Liu, H.-P. Tseng, L.-C. Chen, B.-K. Chen, and W.-C. Chang Functional Cooperation of Simian Virus 40 Promoter Factor 1 and CCAAT/Enhancer-Binding Protein {beta} and {delta} in Lipopolysaccharide-Induced Gene Activation of IL-10 in Mouse Macrophages J. Immunol., July 15, 2003; 171(2): 821 - 828. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Adib-Conquy, P. Moine, K. Asehnoune, A. Edouard, T. Espevik, K. Miyake, C. Werts, and J.-M. Cavaillon Toll-like Receptor-mediated Tumor Necrosis Factor and Interleukin-10 Production Differ during Systemic Inflammation Am. J. Respir. Crit. Care Med., July 15, 2003; 168(2): 158 - 164. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Ziegler-Heitbrock, M. Lotzerich, A. Schaefer, T. Werner, M. Frankenberger, and E. Benkhart IFN-{alpha} Induces the Human IL-10 Gene by Recruiting Both IFN Regulatory Factor 1 and Stat3 J. Immunol., July 1, 2003; 171(1): 285 - 290. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Lefevre, S. Melnik, N. Wilson, A. D. Riggs, and C. Bonifer Developmentally Regulated Recruitment of Transcription Factors and Chromatin Modification Activities to Chicken Lysozyme cis-Regulatory Elements In Vivo Mol. Cell. Biol., June 15, 2003; 23(12): 4386 - 4400. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Goriely, D. Demonte, S. Nizet, D. De Wit, F. Willems, M. Goldman, and C. Van Lint Human IL-12(p35) gene activation involves selective remodeling of a single nucleosome within a region of the promoter containing critical Sp1-binding sites Blood, June 15, 2003; 101(12): 4894 - 4902. [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]
|
|
|
|
 |
|
 M. Jin, H. J. Jung, J. J. Choi, H. Jeon, J. H. Oh, B. Kim, S. S. Shin, J. K. Lee, K. Yoon, and S. Kim Activation of Selective Transcription Factors and Cytokines by Water-Soluble Extract from Lentinus lepideus Experimental Biology and Medicine, June 1, 2003; 228(6): 749 - 758. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Ronni, V. Agarwal, M. Haykinson, M. E. Haberland, G. Cheng, and S. T. Smale Common Interaction Surfaces of the Toll-Like Receptor 4 Cytoplasmic Domain Stimulate Multiple Nuclear Targets Mol. Cell. Biol., April 1, 2003; 23(7): 2543 - 2555. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y.-C. Liao, W.-G. Liang, F.-W. Chen, J.-H. Hsu, J.-J. Yang, and M.-S. Chang IL-19 Induces Production of IL-6 and TNF-{alpha} and Results in Cell Apoptosis Through TNF-{alpha} J. Immunol., October 15, 2002; 169(8): 4288 - 4297. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Tagoh, R. Himes, D. Clarke, P. J.M. Leenen, A. D. Riggs, D. Hume, and C. Bonifer Transcription factor complex formation and chromatin fine structure alterations at the murine c-fms (CSF-1 receptor) locus during maturation of myeloid precursor cells Genes & Dev., July 1, 2002; 16(13): 1721 - 1737. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Taniura, H. Kamitani, T. Watanabe, and T. E. Eling Transcriptional Regulation of Cyclooxygenase-1 by Histone Deacetylase Inhibitors in Normal Human Astrocyte Cells J. Biol. Chem., May 3, 2002; 277(19): 16823 - 16830. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A.-K. Yi, J.-G. Yoon, S.-J. Yeo, S.-C. Hong, B. K. English, and A. M. Krieg Role of Mitogen-Activated Protein Kinases in CpG DNA-Mediated IL-10 and IL-12 Production: Central Role of Extracellular Signal-Regulated Kinase in the Negative Feedback Loop of the CpG DNA-Mediated Th1 Response J. Immunol., May 1, 2002; 168(9): 4711 - 4720. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. R. Hughes, T. S. Tengku-Muhammad, S. A. Irvine, and D. P. Ramji A Novel Role of Sp1 and Sp3 in the Interferon-gamma -mediated Suppression of Macrophage Lipoprotein Lipase Gene Transcription J. Biol. Chem., March 22, 2002; 277(13): 11097 - 11106. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P.-L. Wang and K. Ohura PORPHYROMONAS GINGIVALIS LIPOPOLYSACCHARIDE SIGNALING IN GINGIVAL FIBROBLASTS-CD14 AND TOLL-LIKE RECEPTORS Critical Reviews in Oral Biology & Medicine, March 1, 2002; 13(2): 132 - 142. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Tavor, P. T. Vuong, D. J. Park, A. F. Gombart, A. H. Cohen, and H. P. Koeffler Macrophage functional maturation and cytokine production are impaired in C/EBPepsilon -deficient mice Blood, March 1, 2002; 99(5): 1794 - 1801. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Byrne and D. J. Reen Lipopolysaccharide Induces Rapid Production of IL-10 by Monocytes in the Presence of Apoptotic Neutrophils J. Immunol., February 15, 2002; 168(4): 1968 - 1977. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Wang, W. P. Lafuse, and B. S. Zwilling NF{kappa}B and Sp1 Elements Are Necessary for Maximal Transcription of Toll-like Receptor 2 Induced by Mycobacterium avium J. Immunol., December 15, 2001; 167(12): 6924 - 6932. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Salez, V. Balloy, N. van Rooijen, M. Lebastard, L. Touqui, F. X. McCormack, and M. Chignard Surfactant Protein A Suppresses Lipopolysaccharide-Induced IL-10 Production by Murine Macrophages J. Immunol., May 15, 2001; 166(10): 6376 - 6382. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Frankenberger, B. Passlick, T. Hofer, M. Siebeck, K. L. Maier, and L. H. W. Ziegler-Heitbrock Immunologic Characterization of Normal Human Pleural Macrophages Am. J. Respir. Cell Mol. Biol., September 1, 2000; 23(3): 419 - 426. [Abstract] [Full Text]
|
|
|
|
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W. Ma, W. Lim, K. Gee, S. Aucoin, D. Nandan, M. Kozlowski, F. Diaz-Mitoma, and A. Kumar The p38 Mitogen-activated Kinase Pathway Regulates the Human Interleukin-10 Promoter via the Activation of Sp1 Transcription Factor in Lipopolysaccharide-stimulated Human Macrophages J. Biol. Chem., April 20, 2001; 276(17): 13664 - 13674. [Abstract] [Full Text] [PDF]
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