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The Wistar Institute of Anatomy and Biology, Philadelphia, PA 19104
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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was at the gene transcriptional level and that the
addition of IL-10 to S. aureus- or LPS-treated PBMCs did
not affect mRNA stability. The inhibitory activity of IL-10 was
abrogated by cycloheximide (CHX), suggesting the involvement of a newly
synthesized protein(s). The addition of CHX at 2 h before S.
aureus or LPS also inhibited the accumulation of IL-12 p40 mRNA,
but did not inhibit IL-12 p35 and TNF- mRNA. This finding suggests
that p40 transcription is regulated through a de novo synthesized
protein factor(s), whereas the addition of CHX at 2 h after
S. aureus activation caused superinduction of the
IL-12 p40, IL-12 p35, and TNF-
genes. These results indicate that in human monocytes, the mechanism(s)
of IL-10 suppression of both IL-12 p40 and IL-12
p35 genes is primarily seen at the transcriptional level, and
that the induction of the IL-12 p40 and p35
genes have different requirements for de novo protein synthesis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IL-12 mediates several biologic activities on T and NK cells, including
the induction of IFN-
production, the enhancement of cell-mediated
cytotoxicity, and the costimulation of mitogenesis (5, 6, 7, 8). In addition,
the IL-12 that is produced by accessory cells during early antigenic
stimulation is required for the induction of Th1 responses (9); this
IL-12 is regulated by a positive feedback mechanism mediated by Th1
cells through IFN- or by negative feedback through Th2 cells
secreting IL-10 (10). IL-10 is a potent inhibitor of many of the
functions of monocyte/macrophages, including oxidative burst, nitric
oxide production, phagocytosis, and the production of proinflammatory
cytokines, i.e., IL-12, TNF-, IL-6, and IL-8 (4, 11, 12). Previous
studies of human monocytes demonstrated that IL-10 inhibits the
LPS-stimulated production of inflammatory cytokines by blocking gene
transcription (13, 14). In contrast with these reports, studies of
murine macrophages regarding their activation of TNF- and IL-1 by
LPS show that the down-regulation of mRNA gene expression by IL-10 is
posttranscriptional (15). However, in LPS-activated monocytes, IL-10
was shown to inhibit granulocyte CSF and granulocyte-macrophage CSF
production by destabilizing their mRNA (16). Recently, the mechanism of
IL-10 inhibition of class II MHC expression was shown to be dependent
upon the inhibition of the transport of the class II molecules to the
cell membrane (17). In the present study, we examined the effect of
IL-10 on IL-12 gene regulation in human PBMCs and purified monocytes
stimulated by Staphylococcus aureus or LPS. Our results
demonstrate that IL-10 exerts its suppressive effect on IL-12 p40and p35 as well as TNF- gene expression
mainly at the transcriptional level by a mechanism that requires de
novo protein synthesis. Furthermore, we observe that the LPS- and
S. aureus-induced expression of the IL-12 p40
gene, but not that of IL-12 p35 or TNF genes,
requires de novo protein synthesis, suggesting that the IL-12
p40 gene is regulated differently from the p35 gene and
those genes encoding other proinflammatory cytokines.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Chinese hamster ovary cell-derived human rIL-12
(rhIL-12)4 was provided
by Dr. S. Wolf (Genetics Institute, Cambridge, MA); Chinese hamster
ovary cell-derived rhIL-10 and murine rIL-10 (rmIL-10) was provided by
Dr. K. Moore (DNAX, Palo Alto, CA); and human rTNF- and human
rIFN- were provided by Dr. H.M. Shepard (Genentech, South San
Francisco, CA). The following reagents were purchased from commercial
sources: fixed S. aureus Cowan strain I (Calbiochem-Behring,
La Jolla, CA), LPS (from Escherichia coli, serotype 0127:B8,
Sigma, St. Louis, MO), actinomycin D (Act D) (Calbiochem-Behring),
cycloheximide (CHX) (Sigma), and DMSO (Sigma).
Abs and cytokine assays
The radioimmunoassays (RIAs) for human TNF- and human IFN-
were performed as described previously (18, 19) using mAb pairs
B154.9/B154.7 and B133.1/B133.5, respectively. IL-12 p70 and IL-12 p40
were measured in cell-free supernatants by RIA as described previously
(3) using the mAb pairs 12H4/C8.6 and C11.79/C8.6, respectively. The
RIA for IL-10 was performed using JES3–9D7/JES-12G8 mAbs, which were
kindly provided by Dr. A. O’Garra (DNAX).
Cell preparations
Peripheral blood obtained from healthy donors was anticoagulated with heparin. PBMCs were separated on a Ficoll-Hypaque (Lymphoprep; Nyegaard, Oslo, Norway) density gradient. Monocytes were purified as described previously (20). Briefly, gelatin-coated 175-cm2 tissue culture flasks (Becton Dickinson Labware, Franklin Lakes, NJ) were incubated with autologous plasma for 1 h at 37°C; plasma was aspirated, and monocytes were obtained after a 1-h adherence of the PBMCs to the flasks at 37°C using 10 mM EDTA to remove the cells after carefully rinsing away nonadherent cells. Mycoplasma-free THP-1 human monocytic leukemia cells (21) and murine RAW 264.7 cells (American Type Culture Collection (ATCC), Manassas, VA) were grown in RPMI 1640 medium (Flow Laboratories, Rockville, MD) supplemented with 20% heat-inactivated FBS (Irvine Scientific, Santa Ana, CA). All tissue culture media and supplements were endotoxin-free. Cells were cultured in RPMI 1640 medium supplemented with 10% (20% for THP-1 cells) heat-inactivated FBS in T25 tissue culture flasks (Becton Dickinson Labware) for cytokine and RNA analysis (5 ml, 25 x 106 PBMCs or monocytes/flask; 1 x 107 cell lines/flask), and in T75 cm2 (10 ml, 1 x 108 PBMCs/flask) or T25 flasks (5 ml, 1 x 107 monocytes/flask) for nuclear run-on experiments. Cells were incubated (37°C, 5% CO2) in the presence of the indicated inducers and/or inhibitors following the protocols outlined in the respective figure legends. Cell lines were cultured under the same conditions as the PBMCs and monocytes, except that the cell lines were pretreated with 1.2% DMSO 24 h before the addition of the inducer (22).
Northern blot hybridization
Northern blots were performed as described previously (23).
Briefly, total RNA was extracted from induced and uninduced cells by
the guanidine isothiocyanate method, loaded onto a 1%
agarose-formaldehyde gel (15 µg/lane), and fractionated. IL-12 p40,
TNF-, IL-10, ß-actin, and glyceraldehyde phosphate dehydrogenase
(GAPD) mRNA were detected by sequential hybridization on nylon
membranes (Schleicher & Schuell, Keene, NH) to the respective
[32P]-labeled (Random Primed Kit, Boehringer,
Mannheim, Germany) cDNA fragments. Filters were analyzed on a
PhosphorImager 445S1 (Molecular Dynamics, Sunnyvale, CA); signal
intensities relative to the ß-actin GAPD control were determined
using ImageQuant software (Molecular Dynamics).
RNase protection assay
RNase protection was performed as described previously (4). Briefly, the vector, containing the entire coding region of IL-12 p35, was linearized with the appropriate restriction enzymes and transcribed using [32P]uridine triphosphate (UTP) (800 Ci/mmol; Dupont, Boston, MA) and the riboprobe kit (Promega, Madison, WI) into a complementary RNA (antisense) riboprobe containing a 266-base pair region which was complementary to the sequence in the IL-12 p35. RNA samples (20 µg) were hybridized in solution with an excess of riboprobes (3 x 105 cpm, specific activity 109 cpm/µg, 90°C for 5 min, 42°C for >10 h). A total of 200 µl of RNase solution was added for 30 min at 37°C and the instructions provided for the Ribonuclease Protection Assay RPA II Kit (Ambion, Austin, TX) were followed. The protected fragments were fractionated on 5% polyacrilamide/urea gel and detected using a PhosphorImager 445S1 (Molecular Dynamics).
Nuclear transcription analysis (run-on assay)
The isolation of nuclei and in vitro transcription in the
presence of [32P]UTP (3000 Ci/mmol, Dupont) were
performed essentially as described previously (24, 25). Nuclear RNA was
then isolated after DNase I (Boehringer) and proteinase K (Boehringer)
treatment, followed by four phenol/chloroform/isoamyl alcohol
extractions and ethanol precipitation at -70°C for 2 h.
Unincorporated [32P]UTP was removed using Sephadex G-50
columns (Boehringer). RNA was partially degraded by treatment with 0.2
N NaOH for 10 min at 4°C. [32P]-labeled nuclear RNA was
hybridized for 2 days at 60°C to prehybridized nylon filters
(Schleicher & Schuell) on which 500 ng of denatured PCR-amplified cDNA
corresponding to the coding regions of the IL-12 p40 and
p35, TNF-, and ß-actin genes had
been immobilized using a slot-blot apparatus (Hoeffer Scientific, San
Francisco, CA). After hybridization, filters were washed at room
temperature with 2x SSC, and ssRNA was digested with the same solution
containing 10 µg/ml RNase A at 37°C for 30 min. Filters were then
washed twice in 2x SSC plus 0.1% SDS for 15 min at 50°C and once in
0.1x SSC plus 0.1% SDS for 30 min at 50°C. The extent of
hybridization was quantitated using ImageQuant software on a
PhosphorImager 445S1 (Molecular Dynamics).
Generation of hIL-12 p40 promoter-luciferase stable transfectants
RAW 264.7 cells (ATCC) were electroporated using a Genepulser (Bio-Rad, Hercules, CA) at 350 V and 960 µFD with the 3.3-kilobase (kb) hIL-12 p40 promoter-luciferase construct (26) along with a CMV-neomycin expression vector in a molar ratio of 5:1. At 24 h after electroporation, the cells were plated in 96-well plates at 5000 live cells/well in the presence of 800 fg/ml of G418 (Life Technologies, Grand Island, NY). After a 2-wk period, G418-resistant cells were replated in 96-well plates by limiting dilution to generate single-cell clones. The integration of the construct was verified by PCR with genomic DNA isolated from the clones.
Luciferase assay
Luciferase activity was determined in cell extracts prepared according to the Luciferase Assay Kit (Promega).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-10, and IFN-
secretion in S. aureus-stimulated PBMCs
S. aureus stimulation induces the expression of
multiple cytokines in PBMCs, including IL-12 (p40 and p70), TNF-,
IL-1ß, and IL-10 (4). To determine the temporal appearance of IL-12
relative to that of the other induced cytokines, cell-free supernatant
fluids were collected after 4, 8, 12, and 20 h of S.
aureus stimulation and assessed for cytokine production. S.
aureus-treated PBMCs secreted IL-12 p40, IL-12 p70, TNF-,
IFN-, and IL-10 in a time-dependent manner (Fig. 1
); TNF- production was already
maximal as early as 4 h after S. aureus addition, while
IL-12 p40, IL-12 p70, and IL-10 production reached near maximal levels
at 8 h poststimulation. Induced levels of IFN- were detectable
starting at 8 h and increased up to 20 h poststimulation. At
20 h, the levels of IL-12 p40 were 100-fold higher than those of
IL-12 p70, in agreement with previous reports (27).
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, and IL-10 in S.
aureus- or LPS-stimulated PBMCs or monocytes
To determine the optimal time of cytokine gene expression,
mRNA for IL-12 p40, IL-12 p35, TNF-, and IL-10 were analyzed by
either Northern blot or RNase protection assays at different times
after S. aureus (1/104 wt/v) or LPS (1 µg/ml)
treatment. In PBMCs and monocytes, steady-state levels of IL-12 p40 and
p35 showed similar kinetics of expression and were maximal at 
4 h
after S. aureus stimulation (Fig. 2, A and B).
TNF- mRNA peaked at 4 h after S. aureus induction
and at 1 h after LPS stimulation, followed by a rapid decline
(Fig. 2). The induction of IL-10 mRNA was delayed and more gradual than
that of other genes, peaking between 6 and 12 h after S.
aureus or LPS stimulation (data not shown and Fig. 2B). As previously described (4), when monocytes or
PBMCs were stimulated with S. aureus or LPS in the presence
of added IL-10 (50 U/ml), the cytokine release (data not shown) and
mRNA steady-state levels of IL-12 p40, IL-12 p35, and TNF- (Fig. 2)
were inhibited at all time points, with inhibition ranging from 68 to
90% (Table I). The S.
aureus-induced accumulation of IL-10 mRNA in the presence of IL-10
was not inhibited up to 6 h poststimulation but was completely
blocked by 20 h (Fig. 2B). Studies performed in
the presence of anti-IL-10 demonstrated that endogenous IL-10
inhibited the induction of IL-10 and IL-12 p40 mRNA accumulation after
S. aureus treatment (data not shown), indicating an
autocrine negative feedback control of IL-10. By contrast, LPS-induced
IL-10 mRNA was inhibited by the addition of IL-10 throughout the time
course.
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In a series of experiments, IL-10 was added to the PBMC culture at
different times relative to S. aureus. At 4 h after
S. aureus stimulation, total RNA was extracted and analyzed
by Northern blot hybridization. As shown in Figure 3, S. aureus-induced IL-12 p40
and TNF- mRNA steady-state levels were significantly inhibited when
IL-10 was added over a wide time course. Maximal suppression occurred
when IL-10 was added at 20 h before S. aureus
stimulation. The simultaneous addition of S. aureus and
IL-10 (Fig. 2
) resulted in a significant inhibition of both IL-12 p40
mRNA and TNF- mRNA accumulation. The extent of inhibition remained
the same when the cells were pretreated with IL-10 from 3 h before
until 1 h after the addition of S. aureus and was still
detectable when IL-10 was added after 3 h (Fig. 3 and data not
shown). Because a significant inhibition was observed when IL-10 was
added at 1 h before stimulation, this time point was selected for
additional experiments on the mechanism of inhibition; although a 20-h
treatment resulted in a more complete inhibition, this time point was
not selected, because it was often associated with a loss of viability
in the cultures.
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genes in monocytes and
PBMCs
To determine whether IL-10 inhibits S. aureus- or
LPS-induced gene transcription, nuclear run-on assays were performed
using nuclei isolated from PBMCs or monocytes that had been pretreated
with IL-10 for 1 h followed by S. aureus or LPS
stimulation for 4 h. Figure 4A shows a representative
experiment in which the induction of the transcriptional rate of IL-12
p40 and TNF- induced by S. aureus in monocytes is
significantly inhibited by IL-10. IL-12 p35 gene
transcription appeared to be constitutive with negligible induction,
and the effect of IL-10 was also insignificant (Fig. 4A and
Table II). As shown in Table II, PBMCs
and monocytes treated with S. aureus showed a 14- ± 13-fold
and an 8- ± 3-fold increase in the transcription rates of IL-12
p40 and TNF- genes, respectively, and IL-10
inhibited their transcription by 72 and 59%, respectively. Moreover,
LPS-treated PBMCs and monocytes showed a 13- ± 10-fold and a 3- ±
0.81-fold increase in the transcription rates of IL-12 p40
and TNF- genes, respectively, and IL-10 inhibited their
transcription by 69 and 49%, respectively (Table II). PBMCs cultured
under the same experimental conditions of stimulation as those
described above but pretreated with IFN- for 16 h showed a
significant enhancement of the gene transcriptional level for all three
genes, i.e., IL-12 p40, IL-12 p35, and
TNF- (Fig. 4B). IL-10 inhibited the
gene transcription of IFN--primed S. aureus- or
LPS-induced genes, including IL-12 p35, by 53 to 98%
(Table II).
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mRNA
PBMCs were incubated with or without IL-10 for 1 h, and
S. aureus or LPS were subsequently added. Act D (5 µg/ml)
was added after 4 h to stop further RNA synthesis, and total RNA
was extracted at the times indicated after Act D addition and analyzed
by Northern blotting (Fig. 5). The
calculated t1/2 of IL-12 p40 mRNA and TNF-
mRNA (Fig. 5A) in PBMCs stimulated with S.
aureus was 4 h and 1.8 h, respectively; however, the
t1/2 of p40 was reduced to 2.8 h and that
of TNF- to 0.4 h when LPS was used as an inducer (Fig. 5B). The stability of S. aureus-induced
IL-12 p40 or TNF- mRNA was not altered by treating PBMCs with IL-10
(Fig. 5A). The t1/2 of IL-12
p40 mRNA in LPS-stimulated, IL-10-treated cells (Fig. 5B) was not measurable because of the low IL-12 p40
mRNA steady-state levels.
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We examined the effect of the protein synthesis inhibitor
CHX on the S. aureus- or LPS-induced accumulation of
cytokine mRNA in PBMCs (Fig. 6A), monocytes (Fig. 6B), and THP-1 cells (Fig. 6C). In
these three cell populations, the CHX (10 µg/ml) that was added at
2 h before induction with S. aureus or LPS partially
blocked the induction of IL-12 p40 mRNA, indicating that IL-12 p40
requires de novo protein synthesis for induction. The observation that
IL-12 p35 mRNA accumulation is not inhibited instead is superinduced by
CHX (Fig. 6A) suggests a different mechanism(s) of
regulation for the two components of the heterodimeric IL-12.
Consistent with previous reports (14, 15), the accumulation of TNF-
did not require a newly synthesized protein(s) for induction. Nuclear
run-on experiments in PBMCs treated with CHX at 2 h before
S. aureus stimulation revealed a decreased rate of
transcription of IL-12 p40 but no changes in the
transcription rates of the IL-12 p35 or TNF-
genes (Fig. 7). These results show that
de novo protein synthesis is required for the optimal induction
of IL-12 p40 gene transcription, but that the superinduced,
steady-state mRNA levels of IL-12 p35 and TNF-, observed in
CHX-treated cells, are due to posttranscriptional mechanisms
(28, 29, 30).
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plays a positive feedback
role in IL-12 p40 mRNA induction (4, 31). Therefore, it was important
to determine whether the inhibitory effect of CHX on IL-12
p40 gene expression was a result of the inhibition of IFN-
secretion in the cultures. The addition of neutralizing
anti-IFN- Abs to PBMC cultures at the same time as the CHX
addition and 2 h before S. aureus stimulation showed
that the accumulation of IL-12 p40 mRNA and its inhibition by CHX was
similar in the presence or absence of anti-IFN- Abs, indicating
that the inhibitory effect of CHX on IL-12 p40 induction was not due to
the inhibition of endogenous IFN- secretion (Fig. 8). Furthermore, the addition of
exogenous IFN- could not override the inhibitory effect of CHX (data
not shown).
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CHX was added 2 h before S. aureus stimulation to
determine whether de novo protein synthesis is required for the
inhibitory action of IL-10. Figure 6, A and B
shows a representative experiment performed with PBMCs and monocytes,
respectively, indicating that CHX completely suppresses the ability of
IL-10 to inhibit the S. aureus-induced accumulation of IL-12
p40 and p35 and TNF- mRNA. In nuclear run-on experiments, the
ability of IL-10 to inhibit the transcription of the IL-12
p40 and TNF- genes was similarly suppressed by CHX
(Fig. 7). Interestingly, even when CHX was added after IL-10, i.e, at
the time of or at 2 h after the addition of S. aureus,
a marked suppression of the inhibitory effect of IL-10 on IL-12 p40
mRNA accumulation was observed (Fig. 9).
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In an attempt to search for an in vitro model system whereby the
molecular mechanism(s) of the inhibition of IL-12 gene
expression by IL-10 in monocytic cells could be further investigated,
we stably transfected an hIL-12 p40 promoter-luciferase construct into
the murine monocytic cell line RAW 264.7. This promoter-reporter
construct contains 3.3-kb sequences upstream of the transcription
start site that are linked to a luciferase cDNA, and it has been shown
previously to recapitulate faithfully the regulation of the endogenous
p40 gene in RAW 264.7 cells in response to IFN- and LPS
stimulation (26). G418-resistant clones were generated, and one of the
clones (no. 3) and a mixed population were tested for their response to
IFN-/LPS stimulation as well as to IL-10. As shown in Figure 10 (upper
panels), the endogenous IL-12 p40 production was stimulated
significantly by the combination of IFN- and LPS treatment and was
strongly inhibited by IL-10 added either at 12 h or 2 h
before LPS stimulation in both clone no. 3 and the mixed population.
The stably integrated hIL-12 p40 promoter was also highly inducible by
IFN- and LPS treatment, as measured by luciferase activity, but was
not affected significantly by IL-10 treatment (Fig. 10, lower
panels), suggesting that the transcriptional element(s)
responsible for the inhibition of the IL-12 p40 gene by
IL-10 is not present in this promoter construct.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,
IL-1ß, IL-6, and IL-8 mRNA expression mainly at the level of
transcription with a process depending upon de novo protein synthesis
(14). By contrast, other data suggested that IL-10 acts mainly at the
posttranscriptional level to promote the rapid degradation of TNF-,
IL-1ß (15), and IL-8 (32) mRNA. IL-10 inhibition of IL-12 production
is accompanied by reduced mRNA steady-state levels of the two IL-12
components, p40 and p35 (4). However, it was not known whether this
reduction in IL-12 mRNA accumulation was a consequence of the
inhibition of transcription, an enhancement of mRNA degradation, or
both. Our present results show that IL-10 suppressed S.
aureus- and LPS-induced IL-12 p40 and p35
gene expression mainly at the level of transcription in human PBMCs and
monocytes, without significant modulation of mRNA stability and
regardless of whether they had been primed by IFN-.
Wang et al. (33) suggested that the inhibition of NF
B
activation is an important mechanism for IL-10 suppression of cytokine
gene transcription in human monocytes, while several other
transcriptional factors (e.g., NF-IL-6, activator protein-1, activator
protein-2, glucocorticoid receptor, cAMP response element-binding
protein, Oct-1, and Sp-1) are not affected by IL-10 (33). The
transcriptional factors that induce IL-12 p40 and p35 expression in
monocytes are not defined completely as of yet. The hIL-12 p40 gene
promoter contains a consensus sequence binding site for the ets family
of transcriptional factors, located at -211 to -207, that is critical
to the induction of the promoter by IFN- and LPS in the murine
macrophage cell line RAW 264.7 (26). The extended region in and around
the ets site binds a large nuclear complex, F1, consisting of ets-2,
IFN regulatory factor-1, c-Rel, and a novel 109-kDa protein, which is
absent in unstimulated cells but is highly inducible by IFN- and LPS
(34). An "NFB-half site," originally identified in the mIL-12
p40 promoter (between 121 and -131) (35), is also critical for the
response to IFN- and LPS. In an attempt to identify the element in
the IL-12 p40 promoter that is responsible for IL-10 inhibition, we
tested whether IL-10 could act as a suppressor in transient
transfection experiments of the promoter activity of several constructs
containing up to 3.3 kb of 5' upstream sequences of the IL-12
p40 gene and/or the first intron joined to the luciferase reporter
gene. No inhibition of the transcriptional activity of the IL-12 p40
promoter was ever observed in these experiments (data not shown),
suggesting that the element(s) responsible for IL-10 inhibition may
reside outside the DNA regions analyzed. However, because the
electroporation procedure used in the transient transfection experiment
may have affected the ability of IL-10 to act on the cells, we analyzed
the ability of IL-10 to affect the activity of the 3.3-kb IL-12 p40
promoter in stably transfected RAW 264.7 cells. Again, no inhibitory
activity of mIL-10 was observed in these experiments on the transfected
IL-12 promoter, although IL-10 almost completely suppressed the
expression of the endogenous IL-12 p40 gene in the same
cells.
The S. aureus- or LPS-induced increase in IL-12 p40 mRNA
levels and in p40 gene transcription were nearly abrogated
when cells were pretreated with CHX at 2 h before addition of the
inducer, whereas IL-12 p35 mRNA accumulation was superinduced. Because
CHX did not block the induction of IL-12 p35 or
TNF- transcription by S. aureus or LPS, it is
likely that the transcription of those genes depends upon the
activation of preexisting transcriptional factors, while the decrease
in IL-12 p40 gene expression may be explained by de novo
protein synthesis of a transcriptional factor(s). Although most of the
identified transcriptional factors involved in IL-12 p40
gene transcription are known to be posttranslationally activated, it is
possible that other limiting factors may need to be synthesized de novo
for the induction of IL-12 p40 gene transcription; it is
also possible that the inhibition of the synthesis of these factors is
responsible for the suppressive effect of CHX. Alternatively, CHX may
block the secretion of soluble factors in PBMC cultures, which may
enhance or be required for the optimal induction of IL-12 p40 in
monocytes. In particular, IFN- is known to enhance the induction of
IL-12 p40 by S. aureus or LPS by several-fold (26), and
IFN- produced in PBMC cultures or by contaminant T and NK cells in
enriched monocyte preparations may amplify the expression of the
IL-12 p40 gene. However, it is unlikely that CHX inhibits
IL-12 p40 induction by suppressing IFN- production, as inhibition
was still observed when endogenous IFN- was inhibited by
anti-IFN- Abs and also in the human macrophagic cell line THP-1,
which does not produce IFN-. The requirement for de novo protein
synthesis in the induction of IL-12 p40 gene expression
clearly distinguishes its regulation from that of other proinflammatory
cytokines and from IL-12 p35. Interestingly, the induction of another
phagocytic cell gene activated by LPS, inducible nitric oxide synthase
(iNOS), also requires de novo protein synthesis, and it is blocked by
CHX treatment (36). In this case, the inhibition of protein synthesis
does not block the NFB-dependent binding of nuclear protein in the
macrophage cell line RAW 264.7 but does block the formation of a second
DNA binding complex when a sequence downstream to the iNOS promoter
NFB element is included in the oligonucleotide probe (36). This
sequence contains an NF-IL-6 (C/EBPß) site immediately downstream of
the NFB site (37). Because a C/EBPß site is also present in the
IL-12 p40 promoter downstream of the NFB site, and CAT
enhancer-binding protein ß-deficient mice have been shown to have a
strong impairment of IL-12 production (38), the possibility that a
mechanism similar to that demonstrated for iNOS is involved in the
regulation of IL-12 p40 promoter needs to be investigated.
The superinduction of some cytokine genes, such as
TNF-, IL-1ß (28), IFN-, and
IL-2, (29, 39), is observed when cells are induced in the
absence of CHX for 2 h, followed by the addition of CHX. This
superinduction is attributed to the stabilization of the mRNA, since
earlier studies of the c-fos gene (40) demonstrated that the
mRNA degradation process is dependent upon protein synthesis (28, 29, 30).
In the present study, we showed that, although the induction of IL-12
p40 transcription, unlike that of IL-12 p35 and TNF-, requires de
novo protein synthesis, its steady-state mRNA level could undergo
superinduction if CHX is added 2 h after the inducer, i.e., at a
time when transcription is already activated by S. aureus.
In this respect, the IL-12 p40 gene is similar to other
proinflammatory cytokine genes, including IL-12 p35 and
TNF-.
Our observation, consistent with other reports (14, 15, 32), that the
protein synthesis inhibitor CHX abolished the suppressive effect of
IL-10 on the induction of IL-12 p40, IL-12 p35, and TNF- mRNA,
suggests that IL-10 may exert its negative effect on cytokine gene
transcription through a newly synthesized repressor protein(s). The
inhibition of protein synthesis is unlikely to affect the expression of
IL-10R on the cells, because the ability of IL-10 to inhibit IL-12
expression is immediately recovered upon washing the cells free of CHX
(data not shown). Our data also show that IL-10 acts rapidly to inhibit
IL-12 p40 mRNA accumulation even when added at 1 h postinduction,
and its effect was at least partially prevented by blocking protein
synthesis even when CHX was added at 2 h after the inducer. Thus,
IL-10 is still effective even if added at a time when transcription is
already activated; however, its effect needs to be maintained
throughout the induction period to achieve efficient inhibition. These
findings are not easily reconciled with the demonstration that the
inhibitory effect of IL-10 is at the transcriptional level, and this
effect is likely to be mediated by a second proteinaceous molecule that
is de novo-synthesized upon IL-10 treatment. Furthermore, the nature of
this putative second messenger, as well as the promoter element
responsible for IL-10 inhibition, remains undetermined. Thus, although
this and other studies are starting to shed light on the molecular
mechanism of action of IL-10, many aspects are not yet understood. The
understanding of the reciprocal regulation of IL-12 and IL-10 may
provide some insight into the regulation of cellular and humoral immune
responses and may form the basis for therapeutic intervention.
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Acknowledgments |
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Footnotes |
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2 Current address: Instituto de Biociencias, Universidade Estadual Paulista (UNESP), Botucatu, Brazil.
3 Address correspondence and reprint requests to Dr. Giorgio Trinchieri, Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104.
4 Abbreviations used in this paper: hIL, human IL; mIL, murine IL; Act D, actinomycin D; kb, kilobase; UTP, uridine triphosphate; CHX, cycloheximide; RIA, radioimmunoassay; GAPD, glyceraldehyde phosphate dehydrogenase; iNOS, inducible nitric oxide synthase.
Received for publication July 31, 1997. Accepted for publication February 12, 1998.
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 M. G. Schwacha, C.-S. Chung, A. Ayala, K. I. Bland, and I. H. Chaudry Cyclooxygenase 2-mediated suppression of macrophage interleukin-12 production after thermal injury Am J Physiol Cell Physiol, February 1, 2002; 282(2): C263 - C270. [Abstract] [Full Text] [PDF]
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W. W. S. Kum, S. B. Cameron, R. W. Y. Hung, S. Kalyan, and A. W. Chow Temporal Sequence and Kinetics of Proinflammatory and Anti-Inflammatory Cytokine Secretion Induced by Toxic Shock Syndrome Toxin 1 in Human Peripheral Blood Mononuclear Cells Infect. Immun., December 1, 2001; 69(12): 7544 - 7549. [Abstract] [Full Text] [PDF]
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 K. Qadir, A. Metwali, A. M. Blum, J. Li, D. E. Elliott, and J. V. Weinstock TGF-beta and IL-10 regulation of IFN-gamma produced in Th2-type schistosome granulomas requires IL-12 Am J Physiol Gastrointest Liver Physiol, October 1, 2001; 281(4): G940 - G946. [Abstract] [Full Text] [PDF]
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I. B. McInnes, G. G. Illei, C. L. Danning, C. H. Yarboro, M. Crane, T. Kuroiwa, R. Schlimgen, E. Lee, B. Foster, D. Flemming, et al. IL-10 Improves Skin Disease and Modulates Endothelial Activation and Leukocyte Effector Function in Patients with Psoriatic Arthritis J. Immunol., October 1, 2001; 167(7): 4075 - 4082. [Abstract] [Full Text] [PDF]
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L. Crepaldi, S. Gasperini, J. A. Lapinet, F. Calzetti, C. Pinardi, Y. Liu, S. Zurawski, R. de Waal Malefyt, K. W. Moore, and M. A. Cassatella Up-Regulation of IL-10R1 Expression Is Required to Render Human Neutrophils Fully Responsive to IL-10 J. Immunol., August 15, 2001; 167(4): 2312 - 2322. [Abstract] [Full Text] [PDF]
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M. C. Braun, J. M. Wang, E. Lahey, R. L. Rabin, and B. L. Kelsall Activation of the formyl peptide receptor by the HIV-derived peptide T-20 suppresses interleukin-12 p70 production by human monocytes Blood, June 1, 2001; 97(11): 3531 - 3536. [Abstract] [Full Text] [PDF]
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N. S. Ostlie, P. I. Karachunski, W. Wang, C. Monfardini, M. Kronenberg, and B. M. Conti-Fine Transgenic Expression of IL-10 in T Cells Facilitates Development of Experimental Myasthenia Gravis J. Immunol., April 15, 2001; 166(8): 4853 - 4862. [Abstract] [Full Text] [PDF]
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D. J. Berg, J. Zhang, D. M. Lauricella, and S. A. Moore IL-10 Is a Central Regulator of Cyclooxygenase-2 Expression and Prostaglandin Production J. Immunol., February 15, 2001; 166(4): 2674 - 2680. [Abstract] [Full Text] [PDF]
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L. Malmgaard, S. R. Paludan, S. C. Mogensen, and S. Ellermann-Eriksen Herpes simplex virus type 2 induces secretion of IL-12 by macrophages through a mechanism involving NF-{kappa}B J. Gen. Virol., December 1, 2000; 81(12): 3011 - 3020. [Abstract] [Full Text]
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A. Boonstra, A. van Oudenaren, B. Barendregt, L. An, P. J. M. Leenen, and H. F. J. Savelkoul UVB irradiation modulates systemic immune responses by affecting cytokine production of antigen-presenting cells Int. Immunol., November 1, 2000; 12(11): 1531 - 1538. [Abstract] [Full Text] [PDF]
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X. Ma and L. J. Montaner Proinflammatory response and IL-12 expression in HIV-1 infection J. Leukoc. Biol., September 1, 2000; 68(3): 383 - 390. [Abstract] [Full Text] [PDF]
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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]
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X. Wang, M. Chen, K. P. Wandinger, G. Williams, and S. Dhib-Jalbut IFN-{beta}-1b Inhibits IL-12 Production in Peripheral Blood Mononuclear Cells in an IL-10-Dependent Mechanism: Relevance to IFN-{beta}-1b Therapeutic Effects in Multiple Sclerosis J. Immunol., July 1, 2000; 165(1): 548 - 557. [Abstract] [Full Text] [PDF]
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J. Xiong, K. Kang, L. Liu, Y. Yoshida, K. D. Cooper, and M. A. Ghannoum Candida albicans and Candida krusei Differentially Induce Human Blood Mononuclear Cell Interleukin-12 and Gamma Interferon Production Infect. Immun., May 1, 2000; 68(5): 2464 - 2469. [Abstract] [Full Text] [PDF]
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G. Carra, F. Gerosa, and G. Trinchieri Biosynthesis and Posttranslational Regulation of Human IL-12 J. Immunol., May 1, 2000; 164(9): 4752 - 4761. [Abstract] [Full Text] [PDF]
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 H. Jyonouchi, S. Sun, and F. L. Rimell Cytokine Production by Sinus Lavage, Bronchial Lavage, and Blood Mononuclear Cells in Chronic Rhinosinusitis With or Without Atopy Arch Otolaryngol Head Neck Surg, April 1, 2000; 126(4): 522 - 528. [Abstract] [Full Text] [PDF]
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H. Jyonouchi, S. Sun, C. A. Kennedy, and F. L. Rimell Interferon Gamma Levels in the Sinus, Ear, and Airway in a Rabbit Sinusitis Model Induced by Bacteroides Inoculation Arch Otolaryngol Head Neck Surg, April 1, 2000; 126(4): 529 - 532. [Abstract] [Full Text] [PDF]
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H. Kanamori, S. Krieg, C. Mao, V. A. Di Pippo, S. Wang, D. A. Zajchowski, and D. J. Shapiro Proteinase Inhibitor 9, an Inhibitor of Granzyme B-mediated Apoptosis, Is a Primary Estrogen-inducible Gene in Human Liver Cells J. Biol. Chem., February 25, 2000; 275(8): 5867 - 5873. [Abstract] [Full Text] [PDF]
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X. Ma, J. Sun, E. Papasavvas, H. Riemann, S. Robertson, J. Marshall, R. T. Bailer, A. Moore, R. P. Donnelly, G. Trinchieri, et al. Inhibition of IL-12 Production in Human Monocyte-Derived Macrophages by TNF J. Immunol., February 15, 2000; 164(4): 1722 - 1729. [Abstract] [Full Text] [PDF]
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A. Aicher, G. L. Shu, D. Magaletti, T. Mulvania, A. Pezzutto, A. Craxton, and E. A. Clark Differential Role for p38 Mitogen-Activated Protein Kinase in Regulating CD40-Induced Gene Expression in Dendritic Cells and B Cells J. Immunol., December 1, 1999; 163(11): 5786 - 5795. [Abstract] [Full Text] [PDF]
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J. D. Marshall, J. Chehimi, G. Gri, J. R. Kostman, L. J. Montaner, and G. Trinchieri The Interleukin-12-Mediated Pathway of Immune Events Is Dysfunctional in Human Immunodeficiency Virus-Infected Individuals Blood, August 1, 1999; 94(3): 1003 - 1011. [Abstract] [Full Text] [PDF]
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J. S. Cowdery, N. J. Boerth, L. A. Norian, P. S. Myung, and G. A. Koretzky Differential Regulation of the IL-12 p40 Promoter and of p40 Secretion by CpG DNA and Lipopolysaccharide J. Immunol., June 1, 1999; 162(11): 6770 - 6775. [Abstract] [Full Text] [PDF]
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M. K. Levings and J. W. Schrader IL-4 Inhibits the Production of TNF-{alpha} and IL-12 by STAT6-Dependent and -Independent Mechanisms J. Immunol., May 1, 1999; 162(9): 5224 - 5229. [Abstract] [Full Text] [PDF]
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E. Yamaguchi, J. de Vries, and H. Yssel Differentiation of human single-positive fetal thymocytes in vitro into IL-4- and/or IFN-{gamma}-producing CD4+ and CD8+ T cells Int. Immunol., April 1, 1999; 11(4): 593 - 603. [Abstract] [Full Text] [PDF]
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J. M. Babik, E. Adams, Y. Tone, P. J. Fairchild, M. Tone, and H. Waldmann Expression of Murine IL-12 Is Regulated by Translational Control of the p35 Subunit J. Immunol., April 1, 1999; 162(7): 4069 - 4078. [Abstract] [Full Text] [PDF]
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R. Kishore, J. M. Tebo, M. Kolosov, and T. A. Hamilton Cutting Edge: Clustered AU-Rich Elements Are the Target of IL-10-Mediated mRNA Destabilization in Mouse Macrophages J. Immunol., March 1, 1999; 162(5): 2457 - 2461. [Abstract] [Full Text] [PDF]
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M. C. Braun, J. He, C.-Y. Wu, and B. L. Kelsall Cholera Toxin Suppresses Interleukin (IL)-12 Production and IL-12 Receptor beta 1 and beta 2 Chain Expression J. Exp. Med., February 1, 1999; 189(3): 541 - 552. [Abstract] [Full Text] [PDF]
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M. Delgado, E. J. Munoz-Elias, R. P. Gomariz, and D. Ganea Vasoactive Intestinal Peptide and Pituitary Adenylate Cyclase-Activating Polypeptide Enhance IL-10 Production by Murine Macrophages: In Vitro and In Vivo Studies J. Immunol., February 1, 1999; 162(3): 1707 - 1716. [Abstract] [Full Text] [PDF]
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S. Sanjabi, A. Hoffmann, H.-C. Liou, D. Baltimore, and S. T. Smale Selective requirement for c-Rel during IL-12 P40 gene induction in macrophages PNAS, November 7, 2000; 97(23): 12705 - 12710. [Abstract] [Full Text] [PDF]
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), IL-10 (
), IFN-
); and B,
IL-12 p70 (
). Data from one of four representative experiments
performed with similar results are shown, in which similar kinetics of
production of the cytokines were observed, although the absolute
concentrations of the cytokines produced showed interdonor
variability.
