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Cytokine Research Unit, School of Pathology, Faculty of Medicine, University of New South Wales, Sydney, Australia
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, and TNF in elicited
macrophages. Th2 cytokines generally suppress proinflammatory gene
expression, and IL-4 and IL-13 partially decreased A8 induction in
macrophages and endothelial cells stimulated by LPS or IFN. In
contrast, IL-10 synergized with LPS and IFN to increase mRNA levels
9-fold and secreted A8 levels 
4-fold. IL-10 decreased the optimal
time of mRNA expression induced by LPS from 24 to 8 h. Blocking
experiments indicated that endogenous IL-10 contributes to gene
induction by LPS. Cooperation between IL-10 and LPS was not due to
altered mRNA stability but was dependent on de novo protein synthesis.
Transfection analysis with A8 luciferase constructs confirmed that
synergy was due to increased transcription. The region of the promoter
involved was localized to a 178-bp fragment flanking the transcription
start site of the gene. This region was also responsible for the
suppressive effects of IL-4 and IL-13. Forskolin, CTP-cAMP, and
PGE2 also enhanced LPS- and IFN-induced A8 mRNA, whereas
indomethacin significantly reduced synergy between IL-10 and LPS.
Mitogen-activated protein kinase/cyclooxygenase 2/cAMP pathways
involving CCAAT-enhancing binding protein, located within the active
promoter, may mediate A8 gene up-regulation in a manner mechanistically
distinct to genes regulated by IL-10 via the STAT pathway. A8 exhibits
pleiotropic effects, and the high levels secreted as a result of IL-10
synergy may regulate untoward inflammatory damage by virtue of its an
antioxidant capacity. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-1, and TNF in
elicited Mac (18) and by LPS and IL-1 in microvascular
endothelial cells (MEC; 19) suggested a
proinflammatory role.
Pleiotropic effects of some cytokines are not uncommon, and TGF-
is
a particular example. Like A8, TGF- is chemotactic at picomolar
levels (16), but higher amounts are anti-inflammatory,
and it is a key regulator of wound healing and repair
(20). Both mediators play a role in development (20, 21). Our recent experiments suggest that A8 may be protective
when released at high concentrations, and, in acute inflammatory
responses, it may efficiently scavenge hypochlorite anions produced by
activated neutrophils (22). Neutrophils contain enormous
amounts of A8 (20% of total cytoplasmic protein), which is released
following activation (6). A8 is only expressed in elicited
Mac following activation. LPS- (17), IFN-, and TNF-induced
gene expression is modulated by Ca2+ and by
pathways involving protein kinase (PK)C, leading to activation of
mitogen-activated (MA)PK (18).
In keeping with the negative regulatory role of Th2 cytokines on proinflammatory properties of activated Mac, we investigated the regulation of the A8 gene by IL-4, IL-10, and IL-13. Although Th2 cytokines have some overlapping effects, they also exhibit distinct actions on Mac. For example, IL-10, but not IL-4 or IL-13, down-regulates MHC class II expression and Ag presentation by monocytes (23) and up-regulates monocyte chemoattractant protein (MCP)-1 production by blood monocytes and alveolar Mac (24, 25), although other chemokines are negatively affected (26, 27). Moreover, IL-10 positively regulates several chemoattractant receptors (28, 29, 30). The immunoregulatory mechanisms involving IL-10 appear complex and involve proinflammatory and suppressive properties, although, in general, studies using IL-10-deficient mice indicate that its prime function is to protect the host from overzealous immune/inflammatory responses (31).
Here we present evidence that IL-4 and IL-13 partially suppressed A8 induction in elicited Mac and a microvascular endothelial cell line (MEC). In stark contrast, IL-10 strongly synergized with LPS or IFN to markedly enhance A8 secretion. Synergy may involve a cAMP-dependent pathway, acting principally at the level of transcription, and was confined to a small region of the proximal promoter containing binding sites for Ets and CCAAT/enhancer binding protein (C/EBP) transcription factors.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RPMI 1640 and DMEM were obtained from Life Technologies (Grand
Island, NY), and antibiotics and HBSS were obtained from Sigma (St.
Lois, MO). Bovine calf serum, obtained from HyClone Laboratories
(Logan, UT), was heated at 56°C before use. Plastic flasks and plates
were obtained from Falcon (Lincoln Park, NJ). Thioglycollate (TG) broth
and LPS (Escherichia coli, 055:B5) were obtained from Difco
(Detroit, MI). IFN was obtained from Genentech (San Francisco, CA;
0.032 endotoxin units/mg; specific activity 0.5 x
107 U/mg). Forskolin was obtained from Sigma,
IL-4, IL-10, IL-13, TGF-, and monoclonal anti-mouse IL-10 Ab
from R&D Systems (Minneapolis, MN), and PGE2,
CTP-cAMP, Br-cAMP, Br-cGMP, SB202190, and PD98059 from Calbiochem
(Croydon, Victoria, Australia). For inhibition of mRNA-synthesis, 5
mg/ml actinomycin D (ActD; Calbiochem) in ethanol was diluted into
medium. Cycloheximide (CHX; Sigma) was used as protein synthesis
inhibitor.
Cell culture
TG-elicited Mac were obtained as described (18),
and washed cells (5 x 106) in 60-mm tissue
culture plates were incubated for 1 h at 37°C in 5%
CO2 in air, washed three times with warm (37°C)
HBSS to remove nonadherent cells, and equilibrated in culture medium
for 18 h. Culture medium (3 ml) was replenished before activation,
and populations contained >98% Mac (98% viable by trypan blue
exclusion) and <0.3% neutrophils. Mac were stimulated for up to
96 h with the agents indicated.
The murine monocyte-Mac cell line RAW 264.7 (TIB 71; American Type Culture Collection, Manassas, VA) and the murine edothelioma cell line (MEC) derived from brain (bEND-3) were cultured as described (17, 19). MEC were stimulated once they had reached postconfluence (6–7 days).
Northern analysis
Total cellular RNA (from 5 x 106
Mac) was size fractionated and transferred onto membranes as described
(17). Hybridizations with A8 and A9 riboprobes were for
16 h at 58°C and at 36°C for the 18S rRNA oligoprobe in
formamide-containing buffer as described (19). Membranes
were washed twice at 48°C for 10 min in 2x standard saline citrate
phosphate/EDTA with 0.1% SDS, then twice with 0.1x standard saline
citrate phosphate/EDTA with 0.1% SDS at 65°C for 30 min. Phosphor
imager analyses were performed with a Bio-Rad Molecular Imager GS-525
system (Bio-Rad, Hercules, CA). The relative magnitude of expression
for each gene was determined using software packages and normalized to
the level of 18S RNA on the same blot. Blots were stripped according to
the manufacturer’s instructions.
Quantitation of A8 protein
A8 in supernatants or cell lysates was quantitated using a
double-sandwich ELISA and rabbit polyclonal anti-A8 IgG as
described (7, 17) using recombinant A8 (0.1–50 ng/ml) as
standard. The lower limit of detection was 30 pg/ml.
Reporter plasmids
Truncated promoter fragments of the A8 gene were produced either by using conveniently located restriction endonuclease sites or by nested deletion of a PCR-amplified product. In brief, two fragments extending from EcoRI and XboI sights (-917 and -665, respectively) to an ASP700I site at +465 (8 bp before the start codon in exon 2) were excised from pCP110, end-filled, and placed upstream of the luciferase reporter gene in pGL2-basic vector (Promega, Madison, WI) at an end-filled HindIII site. The constructs were designated pCP-917/+465 and pCP-665/+465, respectively. A 783-bp fragment spanning -316 to +465 was amplified by PCR using pUC/M13 forward sequencing primer (GTTTTCCCAGTCACGAC) and exon primer II (TGTCagatctGATTTCCTTTCAACTGA). PCR was performed as described (32) but using Pfu DNA polymerase (Stratagene, La Jolla, CA). Following BamHI/BglII digestion, the fragment was subcloned into BglII sites of pGL2-basic producing pCP-316/+465. The 5' deletion mutants (with a common 3'-end at +465) generated using the double-stranded Nested Deletion kit (Amersham Pharmacia Biotech, Uppsala, Sweden) yielded the pCP-x/+465 construct series (x represents different 5' ends). The extent of deletion was verified by sequencing.
Transient transfections
RAW 264.7 cells were transiently transfected as described (18) using 2 x 105 cells/well seeded into 12-well plates 24 h before transfection, and then 0.5 µg luciferase reporter plasmid or 0.1 µg reference plasmid (pRL-TK; Promega) was transfected in the presence of DEAE-dextran (Sigma; 300 µg/ml). After 24 h, cells were stimulated for 20 h with the agents indicated, and firefly and Renilla luciferase activities assayed with 20 µl extract using Promega reagents according to the manufacturer’s instructions. Results are expressed as mean ± SD of luciferase activity from three separate experiments. Data was analyzed using the Student t test, and differences were considered statistically significant when p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IFN rapidly and maximally induces A8 mRNA 12 h after
stimulation of elicited Mac, whereas LPS responses are optimal after
24 h (18). To compare the effects of Th2 cytokines on
A8 mRNA expression, TG-elicited Mac were treated with IL-4 and IL-13 in
the presence or absence of IFN or LPS using these time points. IL-4 and
IL-13 did not alter basal A8 mRNA levels (Fig. 1
A) but suppressed positive
responses by 50% (Fig. 1B). Higher doses (30 ng/ml IL-4
or 30 ng/ml IL-13) did not potentiate inhibition (data not shown).
Suppression at the mRNA and protein levels was observed with
LPS-activated RAW 264.7 cells. Secreted A8 produced in response to LPS
was reduced by 90% by IL-4; IL-13 caused 66% suppression (Table I).
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(19).
IL-4, -10, and -13 did not alter basal A8 mRNA levels (Fig. 2A), and IL-4 suppressed the
LPS-induced response by 50%. Although not as potent as IL-4, when
IL-13 was incubated with LPS, gene induction decreased by 20% (Fig. 2B). IL-10 weakly reduced mRNA levels in LPS-activated MEC,
and suppression was additive when IL-10 was mixed with IL-4 or
IL-13.
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amplify inducible A8 in Mac
IL-10 did not induce A8 mRNA directly (Fig. 1
A) but
increased the LPS-activated response >5-fold after 24 h
stimulation, and mRNA levels of IFN plus IL-10-stimulated cells
harvested at 12 h were 2-fold higher (Fig. 1B). Similar
results were observed with RAW 264.7 cells (data not shown) and were
reflected by 3-fold higher A8 in supernatants following LPS plus
IL-10 stimulation (1.78 ± 0.14 ng A8/ml) compared with levels
induced with LPS alone (0.59 ± 0.06 ng/ml; Table I).
TGF- is also involved in resolution of inflammation, and responses
to TGF- were compared with those provoked by the Th2 cytokines. Fig. 1B shows moderate A8 mRNA induction at 24 h, but not at
12 h (Fig. 1A), after addition of TGF-, and
responses to IFN at 12 h and LPS at 24 h increased
1.5-fold when TGF- was included (Fig. 1B). A8 in
supernatants of LPS-activated RAW 264.7 cells increased from 0.59
± 0.06 ng/ml to 0.75 ± 0.26 ng/ml when LPS and TGF- were
cocultured.
Because IL-10 generally down-regulates proinflammatory responses in Mac, the marked up-regulation of the A8 gene is unusual, and mechanisms involved in potentiation of LPS responses were investigated more fully.
Characterization of IL-10-mediated synergy of A8 expression
To confirm synergy between IL-10 and LPS, increasing amounts of
LPS were cultured with a constant amount of IL-10 (10 ng/ml). In
agreement with our previous data (18), elicited Mac
responded to 0.5–50 ng/ml LPS with little potentiation with higher
amounts (500 ng/ml; Fig. 3A).
When IL-10 was included, a linear increase in mRNA was evident with
doses of LPS between 0.5 and 50 ng/ml, with maximal synergy at 50 ng or
greater. An LPS dose of 100 ng/ml was used in all subsequent
experiments.
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B). IL-4
suppressed the synergy, reducing A8 mRNA levels almost to those of LPS
alone (Fig. 3B). A8 in supernatants of RAW 264.7 cells
stimulated with LPS and IL-10 was 1.78 ng/ml, and IL-4 or IL-13 reduced
this to 0.01 ± 0.01 or 0.07 ± 0.02 ng/ml, respectively
(Table I).
IL-10 markedly decreased the optimal time of A8 mRNA induction by LPS.
Low mRNA levels were evident 6 h after addition of LPS; levels
peaked at 24–48 h and declined slowly thereafter (Fig. 4A). The dramatic increases in
mRNA (9-fold) in the presence of IL-10 were maximal by 8–12 h and
declined rapidly to levels below those induced by LPS alone after
48 h. A8 in supernatants reflected this rapid increase and
remained 3- to 4-fold higher than levels in supernatants from Mac
stimulated by LPS alone until 72 h after stimulation (Fig. 4B).
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50% 24 and
48 h after stimulation with LPS (Fig. 4C). Mechanisms of IL-10-mediated A8 gene expression
Many IL-10-mediated effects are via induction of new genes. If
IL-10-increased LPS-induced A8 was due to new protein synthesis,
synergy may be enhanced in Mac pretreated with IL-10 and/or blocked in
Mac costimulated with a protein synthesis inhibitor. A8 mRNA in Mac
preincubated with IL-10 for 1–3 h before addition of LPS were greater
(12-fold increased mRNA levels compared with LPS alone) than
preincubation for 24 h (3-fold increase) (Fig. 5A). When LPS and IL-10 were
added together at the start of the culture, mRNA levels were 10-fold
more than those produced by LPS alone. Synergy was reduced by 60%
when IL-10 was added 3 h after LPS and became less obvious after
later addition. Mac treated with LPS ± IL-10 and a concentration
of CHX which inhibits protein synthesis by >95% (33) did
not express A8 mRNA levels significantly above controls (Fig. 5B), confirming a requirement for new protein synthesis.
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). Although steady-state levels of A8
mRNA were different in LPS plus IL-10- and LPS-treated Mac, degradation
rates were comparable, indicating little effect of IL-10 on the
stability of LPS-induced A8 mRNA.
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A).
IL-10 alone also caused a small but reproducible increase (3-fold),
and, when used with LPS, luciferase activity was potentiated to 14- to
20-fold (Fig. 7A), confirming enhanced transcription.
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A). The minimal construct pCP-178/0 was
used to test whether IL-4 and IL-13 suppression of the LPS-inducible A8
gene was also confined to this region. Fig. 7B shows that
IL-4 and IL-13 reduced LPS-induced responses by 50%. Although IL-4 and
IL-13 did not reduce IL-10-induced luciferase activity, they strongly
suppressed IL-10-LPS synergy, and results correlated closely with
effects on mRNA and protein levels described above. Taken together,
IL-4, IL-13, and IL-10 appear to regulate A8 gene expression of
LPS-stimulated Mac via transcriptionally mediated processes through DNA
elements present in the defined promoter of this gene. Is synergy with IL-10 via PGE2 and cAMP?
Some S100 genes are activated via PKC,- PKA-, and/or
Ca2+-dependent pathways (18, 36, 37). Preliminary experiments to determine intracellular
mechanisms involved in A8 gene regulation by IL-10 were performed.
Although not activating alone, PKA activators PMA, (18),
Br-cAMP, and CTP-cAMP markedly increased IFN- and LPS-induced A8 mRNA,
and the cAMP-elevating agent forskolin potentiated the LPS
response, albeit to lower levels than the other agents (Fig. 8A). CTP-cAMP did not
potentiate A8 mRNA levels induced by LPS plus IL-10.
PGE2 tested at two doses did not alter basal A8
mRNA levels but substantially increased the LPS-induced response (Fig. 8B), whereas indomethacin markedly reduced synergy between
LPS and IL-10 (Fig. 8C).
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C).
In three separate experiments, luciferase activity in transfected Mac
stimulated with LPS alone increased 1.28 ± 0.06-fold with
PGE2 plus LPS (p <
0.05), suggesting a requirement for other elements outside the region
of the essential promoter for full enhancement by
PGE2. The synergy evident with LPS plus IL-10
increased from 1.83 ± 0.08-fold (relative to LPS alone) to
2.34 ± 0.17-fold when PGE2 was included
(p < 0.05). Conversely, indomethacin
suppressed LPS plus IL-10 synergy (p < 0.01,
compared with LPS plus IL-10) to levels provoked by LPS alone, although
LPS-induced luciferase activity was unaffected. Taken together, IL-10
may amplify LPS-induced A8 via PGE2 and cAMP
produced via the cyclooxygenase (COX)-2 pathway. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IL-4 and IL-13 inhibited mRNA induced by LPS in Mac (Fig. 1) and MEC
(Fig. 2) by 50%. Transient transfection analysis of an A8 reporter
construct into RAW 264.7 cells confirmed similar levels of suppression
of gene transcription by both cytokines (Fig. 7B). Although
IL-4 and IL-13 at high levels never reduced mRNA more than 60%,
inhibition was additive when the mediators were used together (Figs. 2 and 7B), yielding secreted protein levels barely above
baseline (Table I). Suppression by IL-4 and IL-13 is not unexpected
because they share the IL-4
receptor component, thereby activating
common signaling pathways (38). In contrast, IL-10
markedly enhanced A8 gene transcription and A8 protein secreted by Mac
cocultured with LPS or IFN (Fig. 1 and Table I). The mRNA levels in MEC
were relatively unaffected (Fig. 2), suggesting cell-specific
regulation of the A8 gene by IL-4, IL-13, and IL-10. In contrast, IL-4
and IL-10 both amplify MCP-1 production by murine MEC (24, 39).
The immunoregulatory roles of IL-10 appear complex and even
paradoxical. In addition to positive effects on Ab production, CTL
development, and growth-costimulatory activity for thymocytes, mast
cells, and B cells (40, 41, 42), IL-10, like IL-4 and IL-13,
inhibits expression of many proinflammatory Mac-derived genes and some
types of inflammation in vivo (31, 43, 44). IL-10 plays a
pivotal role in establishing and maintaining T cell anergy, whereas
IL-4 and IL-13 direct a Th2 cytokine profile typical of an allergic
response or a protective response to parasite infection. Thus,
up-regulation of A8 by IL-10 and suppression by IL-4 and IL-13 suggest
a role for this mediator in resolution of Th1 T cell-mediated immune
responses, particularly because A8 expression by Mac was prolonged and
cAMP mediated (Fig. 8). In contrast, early in an inflammatory response,
IL-10 may regulate leukocyte recruitment by amplifying the A8 gene.
IL-10 affects leukocyte migration in vivo (45, 46), and it
augments plalelet-activating factor receptor, FMLP-R (28)
and CCR-5 (29, 30) expression on monocytes, and their
production of human CC chemokine-4 (47) and MCP-1
chemokines (25) in vitro, strongly supporting a role in
leukocyte migration. Other chemokine genes are either down-regulated or
not affected by IL-10. Unlike the direct positive effect of IL-10 on
chemotactic receptors or human CC chemokine-4 and MCP-1 expression, A8
gene amplification required a positive costimulant, LPS or IFN. TGF-
also has pleiotropic effects and generally suppresses
tissue/inflammatory Mac function but activates monocytes
(48). TGF- directly induced A8 mRNA (Fig. 1A) in elicited Mac and, although not to the same extent as
IL-10, also increased responses provoked by IFN and LPS (Fig. 1B).
Because of the unusual nature and potency of the LPS response to IL-10,
it was examined in more detail. Dose-response experiments (Fig. 3)
confirmed synergy, and the time for optimal A8 mRNA induction by LPS
was reduced from 24–48 h to 8–12 h (Fig. 4A), and secreted
A8 increased 5-fold within 8 h (Fig. 4B). Moreover,
endogenous IL-10, which is up-regulated by LPS in monocytes
(49), contributed significantly to A8 mRNA levels induced
by LPS 24–48 h after stimulation (Fig. 4C). In contrast,
MCP-1 levels are elevated in human monocytes stimulated with LPS in the
presence of anti-IL-10 Ab (25).
Genes induced/enhanced by IL-10 can be regulated by transcriptional
(28) and/or post-transcriptional mechanisms (34, 35). IL-10 abolished the early increase in A8 mRNA levels in
LPS-stimulated Mac occurring soon after addition of ActD to prevent
further transcription (18), suggesting involvement of a
suppressor. After 4 h, the degradation rate of LPS/IL-10 mRNA was
similar to that of Mac stimulated with LPS alone (Fig. 6), indicating
little alteration in mRNA stability. IL-10-mediated mRNA
destabilization in murine Mac occurs through AU-rich elements
(50), and because the 3'-untranslated region of the A8
gene lacks these (18), destabilization by IL-10 is
unlikely. Like A8 mRNA in elicited Mac induced by LPS
(18), the IL-10-mediated increase was dependent on protein
synthesis (Fig. 5), and synergy was greatest in Mac pretreated with
IL-10 for 1–3 h (Fig. 5A), suggesting that IL-10-inducible
factor(s) mediate enhancement.
PGE2 produced by LPS-stimulated Mac
(51) regulates many functions of these cells, including
cAMP and IL-10 generation (52, 53, 54). Suppression of COX-2,
the enzyme at the rate-limiting step of prostanoid production, by
indomethacin indicates that, like endogenous IL-10, endogenous
PGE2 contributed to A8 mRNA levels 24 h
after stimulation of Mac with LPS or LPS/IL-10 (Fig. 8B).
PGE2 and cAMP analogs alone did not initiate or
weakly initiated A8 gene expression (Fig. 8A). Reporter
assays confirmed low transcriptional activity provoked directly by
CTP-cAMP in RAW 264.7 cells (Fig. 8C).
PGE2 and CTP-cAMP, but not cGMP, analogs (data
not shown) strongly amplified LPS-and IFN-stimulated responses (Fig. 8, A and B) and increased transcription of the
luciferase reporter in the presence of LPS or LPS/IL-10 (Fig. 8C). These studies suggest indirect regulation of the A8
gene in macrophages by LPS, involving LPS-mediated production of IL-10
and PGE2, via a cAMP-dependent pathway.
The high A8 mRNA and protein levels induced by LPS plus IL-10 were
markedly reduced by IL-4 and IL-13 (Fig. 3B and Table I),
confirming their important negative regulatory role. Although
mechansims are unclear, IL-4 and IL-13 decreased transcription of the
minimal A8 promoter in RAW 264.7 cells stimulated with LPS/IL-10 by
60% (Fig. 7B). Constitutive expression of A8 protein in
human monocytes is suppressed by IL-4 and IL-10, and decreases are also
greatest with the mediators combined (55). Reduced
production of IL-10 and PGE2 by IL-4 and IL-13,
as occurs with some monocyte/Mac populations (56, 57, 58), may
contribute to suppression.
The 178-bp region of the A8 promoter is necessary and sufficient for
gene induction by LPS, and transient transfection experiments confirmed
the involvement of this region in LPS synergy with IL-10 (Fig. 7A). IL-10R signaling involves activation of Jak kinases and
phosphorylation of receptor docking sites for members of the STAT
family of transcription factors (59, 60). IL-10-mediated
potentiation can occur via binding of phosphorylated STAT1 and STAT3
multimeric complexes to the IFN- response region, such as in the
FcRI gene promoter (61). However, database searches
indicate no STAT binding elements or IFN- response region in the
essential A8 promoter, although several copies of Ets, E-Box and
C/EBP consensus sequences were located. C/EBP is strongly associated
with cAMP signaling pathways, and its expression is intensified by cAMP
(62, 63, 64). C/EBP is conserved in the promoter regions of
the murine and human A8 genes and is located within an enhancer element
in the human A9 gene (65, 66). The human A8 promoter is
activated by C/EBP (67). C/EBP can be up-regulated
following monocyte activation (68) and is involved in
induction of the COX-2 gene in LPS-activated RAW 264.7 cells
(69), suggesting a mechanism for a potentiating feedback
loop in A8 gene regulation in Mac by PGE2. IL-4
and IL-13 decrease TNF--induced C/EBP in synovial fibroblasts,
whereas IL-10 up-regulates basal levels (70), suggesting a
common mechanism. Examination of the role of this transcription factor
in A8 gene regulation by pro- and anti-inflammatory cytokines is
underway in our laboratory.
Induction of A8 by LPS is dependent on multiple signals, including changes in intracellular calcium, PKC, and activation of MAPK (18), and early induction of A8 mRNA by LPS/IL-10 (4–8 h) was abolished by the MAPK inhibitors SB202190 and PD98059 (data not shown). Here we show that IL-10, COX-2, and cAMP contribute to the LPS response, and we propose that the MAPK/COX-2/cAMP pathways involving C/EBP may regulate transcriptional synergy between LPS and IL-10 in a manner similar to up-regulation of the arginase gene by LPS and IL-10 (71, 72, 73, 74). This differs mechanistically to genes regulated by IL-10 via the STAT pathway. Multiple factors may be involved and may include a newly synthesized protein that regulates release of a suppressor to promote rapid transcription.
A8 may exhibit pleiotropic effects, and the high levels secreted as a result of IL-10 synergy may regulate untoward inflammatory damage by virtue of A8’s capacity to act as an antioxidant (22), thereby protecting against acute cytokine-mediated pathology.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Carolyn Geczy, School of Pathology, University of New South Wales, Sydney, Australia.
3 Abbreviations used in this paper: A8, S100A8; A9, S100A9; Mac, macrophages; MEC, microvascular endothelial cells; PK, protein kinase; MA, mitogen-activated; MCP, monocyte chemotactic protein; C/EBP, CCAAT/enhancer binding protein; TG, thioglycollate; ActD, actinomycin D; CHX, cytoheximide; COX-2, cyclooxy-genase 2.
Received for publication December 20, 2000. Accepted for publication March 15, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
2 integrin Mac-1 on neutrophils. J. Immunol. 160:1427. and TNF regulate macrophage expression of the chemotactic S100 protein S100A8. J. Immunol. 164:4916.. Annu. Rev. Immunol. 16:137.[Medline]
or IL-10. J. Immunol. 151:6370.[Abstract]
selectively inhibits lipopolysaccharide-inducible JE/monocyte chemoattractant protein-1 and KC/GRO/melanoma growth-stimulating activity gene expression in mouse peritoneal macrophages. J. Immunol. 153:2204.[Abstract]
, interleukin-4, and interleukin-10. Ann. NY Acad. Sci. 685:713.[Medline]
interferon, and interleukin-10 mRNA production in murine macrophages. Infect. Immun. 65:3239.[Abstract]
, TNF- and PGE2 production by human peritoneal macrophages. Immunology 72:344.[Medline]
RI/CD64) gene and STAT protein binding to the IFN- response region (GRR) are regulated differentially in human neutrophils and monocytes by IL-10. J. Immunol. 160:911. as a cyclic AMP-responsive nuclear regulator. J. Biol. Chem. 273:14950. in the thyroid hormone and cAMP induction of phosphoenolpyruvate carboxykinase gene transcription. J. Biol. Chem. 274:211. regulate the monocytic/myeloic gene MRP14. Immunobiology 199:148.[Medline]
and C/EBP isoform expression during the lipopolysaccharide-mediated acute-phase response. Mol. Cell. Biol. 16:2295.[Abstract]
-induced prostaglandin E2 production by the antiinflammatory cytokines interleukin-4, interleukin- 10, and interleukin-13 in osteoarthritic synovial fibroblasts: distinct targeting in the signaling pathways. Arthritis Rheum. 42:710.[Medline]
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 J. Vakkila, R. Jaffe, M. Michelow, and M. T. Lotze Pediatric cancers are infiltrated predominantly by macrophages and contain a paucity of dendritic cells: a major nosologic difference with adult tumors. Clin. Cancer Res., April 1, 2006; 12(7): 2049 - 2054. [Abstract] [Full Text] [PDF]
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 M. Eikmans, M. C. Roos-van Groningen, Y. W.J. Sijpkens, J. Ehrchen, J. Roth, H. J. Baelde, I. M. Bajema, J. W. de Fijter, E. de Heer, and J. A. Bruijn Expression of Surfactant Protein-C, S100A8, S100A9, and B Cell Markers in Renal Allografts: Investigation of the Prognostic Value J. Am. Soc. Nephrol., December 1, 2005; 16(12): 3771 - 3786. [Abstract] [Full Text] [PDF]
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K. Hsu, R. J. Passey, Y. Endoh, F. Rahimi, P. Youssef, T. Yen, and C. L. Geczy Regulation of S100A8 by Glucocorticoids J. Immunol., February 15, 2005; 174(4): 2318 - 2326. [Abstract] [Full Text] [PDF]
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G. M. Anstead, B. Chandrasekar, Q. Zhang, and P. C. Melby Multinutrient undernutrition dysregulates the resident macrophage proinflammatory cytokine network, nuclear factor-{kappa}B activation, and nitric oxide production J. Leukoc. Biol., December 1, 2003; 74(6): 982 - 991. [Abstract] [Full Text]
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K. Vandal, P. Rouleau, A. Boivin, C. Ryckman, M. Talbot, and P. A. Tessier Blockade of S100A8 and S100A9 Suppresses Neutrophil Migration in Response to Lipopolysaccharide J. Immunol., September 1, 2003; 171(5): 2602 - 2609. [Abstract] [Full Text] [PDF]
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C. Ryckman, K. Vandal, P. Rouleau, M. Talbot, and P. A. Tessier Proinflammatory Activities of S100: Proteins S100A8, S100A9, and S100A8/A9 Induce Neutrophil Chemotaxis and Adhesion J. Immunol., March 15, 2003; 170(6): 3233 - 3242. [Abstract] [Full Text] [PDF]
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A.-F. Petit-Bertron, C. Fitting, J.-M. Cavaillon, and M. Adib-Conquy Adherence influences monocyte responsiveness to interleukin-10 J. Leukoc. Biol., January 1, 2003; 73(1): 145 - 154. [Abstract] [Full Text] [PDF]
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L. Williams, G. Jarai, A. Smith, and P. Finan IL-10 expression profiling in human monocytes J. Leukoc. Biol., October 1, 2002; 72(4): 800 - 809. [Abstract] [Full Text] [PDF]
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C. Ryckman, G. A. Robichaud, J. Roy, R. Cantin, M. J. Tremblay, and P. A. Tessier HIV-1 Transcription and Virus Production Are Both Accentuated by the Proinflammatory Myeloid-Related Proteins in Human CD4+ T Lymphocytes J. Immunol., September 15, 2002; 169(6): 3307 - 3313. [Abstract] [Full Text] [PDF]
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 M. J. Raftery, Z. Yang, S. M. Valenzuela, and C. L. Geczy Novel Intra- and Inter-molecular Sulfinamide Bonds in S100A8 Produced by Hypochlorite Oxidation J. Biol. Chem., August 31, 2001; 276(36): 33393 - 33401. [Abstract] [Full Text] [PDF]
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) or stimulated with 10 ng/ml IL-10 (
), 100 ng/ml LPS
(
), or IL-10 and LPS combined (•) for the times indicated before
Northern blot analysis. Relative A8 mRNA levels are presented as fold
increases of maximal mRNA levels induced by LPS. Results are
representative of two experiments. B, A8 in supernatants
of elicited Mac, untreated (
). IL-10 (
; 10 ng/ml) was added at the indicated times relative
to the addition of LPS (
)-treated Mac are plotted against time after ActD
addition. Insert shows results of Northern blot analysis of A8 mRNA.
Results are representative of three experiments.
