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* Department of Pathology, Section of General Pathology, University of Verona, Verona, Italy; and
Molecular and Cellular Immunology, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-6, and GM-CSF secretion in response to
LPS at levels similar to those exerted by IL-10 in LPS-stimulated
wild-type J774. Constitutive SOCS-3 expression also down-regulated the
mRNA expression of inducible NO synthase and IL-6 and impaired the
production of TNF-, mainly at a post-transcriptional level. In
addition, SOCS-3-transfected cells displayed a constitutive expression
of the IL-1R antagonist gene, consistent with the observation that
IL-10 enhances IL-1R antagonist mRNA in LPS-stimulated wild-type cells.
Furthermore, in peritoneal macrophages harvested from mice carrying
heterozygous disruption of the SOCS-3 gene, IL-10 was less effective in
repressing LPS-stimulated TNF- and NO production. Taken together,
our data show that SOCS-3 inhibits LPS-induced macrophage activation,
strongly supporting the idea that it plays a role in the molecular
mechanism by which IL-10 down-modulates the effector functions of
LPS-activated macrophages. Finally, we show that forced expression of
SOCS-3 significantly suppresses the ability of IL-10 to trigger
tyrosine phosphorylation of STAT3. Therefore, SOCS-3 functions both as
an LPS signal inhibitor and as a negative feedback regulator of
IL-10/STAT3 signaling. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-induced MHC class II expression, reactive oxygen intermediates
and NO production, phagocytosis, and synthesis of proinflammatory
cytokines in response to LPS or other agonists (8, 9). The intracellular mechanisms by which IL-10 inhibits proinflammatory cytokine production by LPS-activated macrophages remain largely unclear, even though several studies have shown that they are dependent on de novo protein synthesis (10, 11). In this context it has been recently reported that one of the IL-10 target genes is suppressor of cytokine signaling 3 (SOCS-3)3 (12, 13). The latter protein is a member of a growing family of suppressor of cytokine signaling molecules that were initially described as feedback inhibitors of cytokine signaling pathways and have subsequently gained a widespread role in the negative cross-talk between cytokines and other proinflammatory stimuli (14). A number of studies have demonstrated that SOCS-3 mRNA can be induced by different classes of agonists, including cytokines, hormones, and infectious agents in different tissues (15). The observations that the inhibitory actions of IL-10 and IL-10-mediated induction of SOCS-3 mRNA are temporally associated (12, 13) indicate that SOCS-3 may be a cytoplasmic effector molecule of IL-10-mediated inhibitory mechanisms (16).
To determine whether the mechanisms by which IL-10 negatively regulates
the proinflammatory responses of macrophages to LPS occur via SOCS-3
protein expression, we generated a macrophage cell line stably
expressing SOCS-3 cDNA. In this work we show that in SOCS-3-transfected
macrophages the production of LPS-induced NO, TNF-, IL-6, and GM-CSF
is inhibited to an extent similar to that observed in wild-type cells
treated with IL-10 and LPS. In contrast, expression of the IL-1R
antagonist (IL-1ra) gene in SOCS-3-transfected macrophages is
constitutive and remained inducible by LPS. We also show that the
inhibitory activities of IL-10 on LPS-induced NO and TNF- production
are severely impaired in peritoneal macrophages isolated from mice
carrying heterozygous disruption of the SOCS-3 gene. Finally, we show
that SOCS-3 constitutive expression almost completely blocks
IL-10-induced STAT3 tyrosine phosphorylation. Collectively, our data
suggest that SOCS-3 may act as a negative feedback regulator and as an
intracellular mediator used by IL-10 to exert its anti-inflammatory
actions.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The mouse macrophage cell line J774 (provided by Dr. V. Kruys,
Université Libre de Brussels, Brussels, Belgium) was maintained
in DMEM (BioWhittaker, Verviers, Belgium) supplemented with 5% low
endotoxin FBS (Biochrom Seromed, Berlin, Germany), and passaged twice
weekly. Cells (8 x 105 cells/well) were
seeded into six-well plates and transfected 24 h later with the
pcDNA3-myc-SOCS-3 construct (17) using the
Superfect transfection reagent (Qiagen, Valencia, CA), according to the
manufacturer’s instructions. Stably transfected cells were selected 3
days after transfection by addition of 0.3 mg/ml Geneticin (Life
Technologies, Paisley, U.K.). These concentrations were maintained for
the first 3 days and increased every third day up to 0.7 mg/ml.
Twenty-four clones were then isolated and cultured in the presence of
0.7 mg/ml Geneticin. In all the experiments cells were seeded in tissue
culture plates and stimulated 24 h later with 100 ng/ml LPS
(from Escherichia coli serotype 026:B6; Sigma-Aldrich, St.
Louis, MO) in the presence or the absence of 20 ng/ml murine IL-10
(Immunokontact, Abingdon, U.K.). In selected experiments cell were also
stimulated with 10 ng/ml murine IFN- (PeproTech, London, U.K.) or 10
ng/ml murine IL-6 (Immunokontact). All reagents used were of the
highest available grade and were dissolved in pyrogen-free water for
clinical use.
Primary mouse macrophages isolation and culture
Primary resident macrophages were isolated by peritoneal lavages
with 10 ml RPMI 1640 containing 10% FBS from 6- to 8-wk-old
SOCS3+/- mice (18) backcrossed onto
a C57BL/6 background and from their wild-type
(SOCS3+/+) littermates. Cells were seeded at a
density of 
1 x 106 cells/well in 24-well
tissue culture plates and were cultured overnight in RPMI 1640 plus
10% FBS at 37°C in a humidified incubator containing 5%
CO2. The following day, all nonadherent cells
were removed by washing with PBS. Adherent primary macrophages were
then stimulated with LPS (100 ng/ml) in the presence or the absence of
20 ng/ml IL-10 for 3–12 h, and the production of NO and TNF- was
determined as described below.
Total RNA extraction and RNase protection assay (RPA) analysis
After stimulation with LPS and/or IL-10 or IFN-, cells were
harvested, and total RNA was extracted using a commercial kit (SV total
RNA isolation system; Promega, Madison, WI). Analysis of the expression
of different genes was conducted at once by RPA, using the RiboQuant
Custom Mouse Probe Set containing probes for iNOS, TNF-, IL-1Ra,
IL-6, L32, and GAPDH (BD Pharmingen, La Jolla, CA) according to the
manufacturer’s instructions. Expression levels of the various genes
were quantified by Instantimager analysis (Packard Instruments, Palo
Alto, CA).
Western blots
Preparation of cell lysates and immunoblot analysis were conducted as previously described (19) using the following primary Abs: anti-NH2 terminus SOCS-3 (Immuno-Biological Laboratories, Tokyo, Japan) diluted at 5 µg/ml; anti-Y705-P STAT3, anti-T202/Y204-P extracellular signal-regulated kinase 1/2 (ERK1/2) mitogen-activated protein kinase (MAPK), anti-T180/Y182-P p38 MAPK, anti-T183/Y185-P stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) MAPK, anti-p38 MAPK, and anti-SAPK/JNK MAPK (New England Biolabs, Beverly, MA) diluted as recommended by the manufacturer; and anti-STAT3 diluted at 1/2000, anti-ERK1 and anti-ERK2 diluted 1/1000 (Santa Cruz Biotechnology, Santa Cruz, CA), and anti-iNOS (Transduction Laboratories, Lexington, KY) diluted at 1/500. Ab binding was detected by using HRP-conjugated anti-mouse or anti-rabbit IgG (1/4000 dilution in TBS-Tween) and was revealed using the chemiluminescence system (ECL; Amersham Pharmacia Biotech, Arlington Heights, IL).
Detection of cytokine and nitrite secretion
The extracellular release of cytokines and nitrite was assayed
in cell-free supernatants collected after cell stimulation for the
indicated times. Cytokine levels in cell-free supernatants of J774
cells and primary macrophages were determined using commercial ELISA
kits for mouse TNF- (Minikit; Endogen, Woburn, MA), mouse
GM-CSF (Quantikine Immunoassay; R&D Systems, Minneapolis, MN), and
mouse IL-6 (Quantikine). The production of NO was determined by
assaying culture supernatants for
NO2- accumulation by the Griess
reagents (20).
Statistical analysis
Data are expressed as the mean ± SE. Statistical evaluation was performed using Student’s t test and was considered significant at p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To clarify whether IL-10-induced SOCS-3 acts as a key mediator of
IL-10 anti-inflammatory responses, we stably transfected Myc-tagged
SOCS-3 cDNA into the mouse macrophage cell line J774. Our preliminary
experiments demonstrated that in these cells IL-10 not only strongly
inhibits LPS-induced NO and TNF- production, but also induces SOCS-3
mRNA and protein expression. Three weeks after antibiotic selection,
transfectants were cloned, and expression levels of SOCS-3 in
individual clones (named JS) were verified by immunoblot analysis,
using anti-SOCS-3 Abs. Albeit at variable levels, all the clones
isolated constitutively expressed a protein of the predicted molecular
mass of Myc-SOCS-3, but only four of them (JS#10, JS#11, JS#L, and
JS#13) displayed levels of exogenous myc-SOCS-3 expression
similar to those of endogenous SOCS-3 induced by IL-10 plus LPS in
parental J774 cells (Fig. 1
). We
therefore chose these four clones for subsequent analysis of LPS
responsiveness.
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Fig. 2 shows that in J774
macrophages LPS-induced iNOS mRNA accumulation is detectable as early
as after 2 h of incubation, peaks at 6 h, and gradually
decreases over 24 h (not shown). In the presence of IL-10,
LPS-induced iNOS mRNA expression is inhibited by 45% after 2 h
and by 69% after 6 h. Similarly, the levels of iNOS mRNA induced
by LPS in SOCS-3-expressing clones resulted lower compared with those
in LPS-stimulated J774 parental cells. The degree of inhibition of
LPS-induced iNOS mRNA expression in the different clones was variable,
corresponding at 2 h to 69% in JS#11, 85% in JS#L, 48% in
JS#10, and 54% in JS#13. In contrast, iNOS mRNA steady state levels
induced by LPS plus IFN- was not modified in SOCS-3-expressing
clones compared with parental J774.
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A).
Under these conditions, LPS-induced iNOS protein in J774 cells was
already detectable after 3 h and continued to accumulate up to
24 h, but was strongly diminished if IL-10 was present in the
culture medium (Fig. 3A). Similarly, and in line with RPA
analysis, the synthesis of iNOS protein induced by LPS in JS clones was
lower than that induced in J774 (Fig. 3A). As a result of
the reduced levels of LPS-induced iNOS enzyme observed either in J774
treated with IL-10 or in SOCS-3-expressing JS clones, the production of
NO was, accordingly, decreased. Addition of IL-10 to parental J774 in
fact reduced LPS-induced NO production by an average of 61 ± 5%
(n = 6) at 24 h. Similarly, NO production in
response to LPS was reduced in JS clones compared with control J774 by
an average of 82 ± 4% in clone JS#11 (n = 8),
51 ± 7% in clone JS#13 (n = 2), and 88 ±
7% in clone JS#L (n = 5; Fig. 3B). However,
NO production triggered by IFN- in combination with LPS was not
significantly inhibited by either IL-10 in parental J774 (not shown) or
forced SOCS-3 expression (Fig. 3B).
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expression and production
Because IL-10 is well known to suppress cytokine production in
many cellular systems, including J774 (21, 22), in
subsequent experiments we investigated the effect of forced SOCS-3
expression on LPS-induced TNF-. In agreement with previous studies,
addition of IL-10 resulted in a strong inhibition of LPS-induced
TNF- production in J774 cells (Fig. 4A), which was, on the
average, 48 ± 10% at 3 h, 80 ± 8% at 6 h, and
80 ± 13% at 24 h (n = 4; Fig. 4B). Similarly, stable expression of SOCS-3 reduced TNF-
production in response to LPS by an average of 70 ± 15% at
3 h, 85 ± 5% at 6 h, and 52 ± 18% at 24 h
in clone JS#11 (n = 4) and by an average of 88 ±
6% at 6 h and 77 ± 4% at 24 h in clone JS#10
(n = 5; Fig. 4, A and B).
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production by activated JS
clones reflected a poor induction of TNF- transcripts by LPS, we
quantified the amounts of TNF- mRNA following LPS stimulation in the
presence or the absence of IL-10 in parental J774 cells or in
SOCS-3-expressing clones. As shown in Fig. 2
, TNF- mRNA accumulation
was rapidly induced by LPS in wild-type J774, peaking at 2 h and
slightly decreasing within 6 h. Differently from the effect on
iNOS mRNA induction, IL-10 inhibited LPS-induced TNF- mRNA
accumulation by only 17 ± 8% at 2 h and 29 ± 11% at
6 h (Fig. 4B, average of three independent
experiments). In a similar manner the levels of LPS-induced TNF-
transcripts in clones JS#11 and JS#L were not significantly different
from those observed in parental J774 cells (Fig. 2) and were only
slightly reduced in clones JS#10 (23% inhibition at 2 h) and
JS#13 (13% inhibition at 2 h; Figs. 2 and 4B).
Overall, these data indicate that IL-10 as well as SOCS-3 constitutive
expression inhibit macrophage production of TNF- primarily at a
post-transcriptional level. Effect of SOCS-3 on LPS-induced IL-6 and GM-CSF production
Analysis of the effects of SOCS-3 forced expression was then
extended to LPS-induced IL-6 gene expression and protein secretion as
well as to LPS-induced GM-CSF production. As shown in the RPA of Fig. 2, up-regulation of IL-6 mRNA by LPS peaked at 6 h and was
strongly suppressed by IL-10. In a similar manner LPS failed to induce
IL-6 gene expression in SOCS-3-expressing clones (Fig. 2). In contrast
with iNOS and TNF-, IL-6 transcripts induced by LPS plus IFN- was
inhibited in SOCS-3-expressing clones, indicating that the expression
of IL-6 mRNA is regulated differently from that of iNOS or TNF-. In
line with the results obtained at the mRNA level, the production of
IL-6 induced by LPS was dramatically inhibited by IL-10 in J774 cells
and by constitutive expression of SOCS-3 in JS clones (Table I). Under the same experimental
conditions the production of GM-CSF stimulated by LPS was strongly
inhibited by IL-10 in J774 and was totally blocked in JS clones
(Table I).
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IL-10 exerts its anti-inflammatory actions not only by
inhibiting the production of proinflammatory mediators, but also by
promoting the synthesis and release of natural inhibitors of certain
proinflammatory cytokines (8). According to previous
findings (23), Fig. 5 shows
that in parental J774 cells the IL-1ra gene is strongly up-regulated in
response to LPS and is further enhanced in cells costimulated with
IL-10. Remarkably, RPA analysis demonstrates that there is a slight
increase in the basal levels of IL-1ra gene expression in all JS clones
(Fig. 5). However, in contrast with the inhibitory effect of SOCS-3
forced expression on endotoxin stimulation of proinflammatory
mediators, addition of LPS was still effective in increasing IL-1ra
mRNA expression in JS clones.
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Activation of ERK1/2, SAPK/JNK, and p38 MAPK as well as NF-
B
has been reported to be involved in the signaling cascade(s) mediating
LPS-induced TNF- production (24). To investigate
whether IL-10 treatment or SOCS-3 constitutive expression inhibits
LPS-induced TNF- production by interfering with MAPK/SAPK signaling,
we examined the effects of IL-10 and SOCS-3 transfection on the
LPS-induced activation of ERK1/2, SAPK/JNK, and p38 MAPK using
phospho-specific Abs (Fig. 6). Fig. 6A shows that maximal activation of p38 by LPS is achieved
within 2 min, remains stable over 15 min, and decreases thereafter (not
shown), whereas optimal activation of ERK1/2 and SAPK/JNK in wild-type
J774 is achieved after 30 min of LPS stimulation and remains unchanged
at 60 min (Fig. 6B). In these experiments neither IL-10 in
wild-type J774 (Fig. 6, A and B) nor SOCS-3
expression in JS clones #10 (Fig. 6, A and B) and
#11 (not shown) influenced either the levels or the kinetics of p38
MAPK, ERK1/2, and SAPK/JNK activation in LPS-stimulated cells. The
apparent decrease in SAPK/JNK phosphorylation in J774 in the presence
of IL-10 and in JS clones was due to the slightly reduced levels of
SAPK/JNK proteins in these cells (Fig. 6B). Similarly,
LPS-induced NF-B activation was unaffected by either IL-10 or SOCS-3
expression in J774 cells (data not shown).
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To provide more evidence on the capacity of SOCS-3 to mediate the
inhibitory effects of IL-10, we monitored whether IL-10 was able to
inhibit LPS-induced TNF- and NO production in primary peritoneal
macrophages isolated from SOCS-3+/- mice
(18) backcrossed onto a C57BL/6 background and from their
wild-type (SOCS-3+/+) littermates. As depicted in
Fig. 7, A and B,
showing representative experiments, IL-10 inhibited the production of
TNF- and the release of NO from LPS-activated macrophages harvested
from wild-type mice by 69.7 ± 8.9% (n = 3; Fig. 7A) and 71.5 ± 2.8% (n = 3; Fig. 7B), respectively. In contrast, the ability of IL-10 to
inhibit LPS-induced TNF- and NO production in macrophages derived
from SOCS-3+/- mice decreased to 20.3 ±
3.7% (Fig. 7A), and to 31 ± 0.9% (Fig. 7B), respectively. The reduced inhibitory activity of IL-10
on LPS-induced TNF- and NO production in
SOCS-3+/- cells is significantly different from
that in SOCS-3+/+ cells
(p = 0.001). In parallel, Western blot analysis
revealed that LPS plus IL-10-induced SOCS-3 protein expression was
substantially reduced in adherent macrophages from
SOCS-3+/- mice compared with macrophages from
SOCS-3+/+ mice (Fig. 7C), indicating
that SOCS-3 is required for mediating the anti-inflammatory action
of IL-10.
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The activation of STAT3 has been shown to play a key role in IL-10
signal transduction (8). In contrast, SOCS-3 has been
described to target the STAT pathway activated by different cytokines
(14). We therefore examined whether activation of STAT3 in
response to IL-10 was impaired in SOCS-3-expressing clones. As shown in
Fig. 8A, IL-10 promotes a
strong and time-dependent STAT3 tyrosine phosphorylation in J774 cells
that is, however, strongly inhibited in JS#11 and JS#13 cells (Fig. 8A), even if the cytokine is used at doses up to 100 ng/ml
(Fig. 8B). STAT3 tyrosine phosphorylation induced by IL-6
was also substantially inhibited by SOCS-3 constitutive expression
(Fig. 8C), confirming the results obtained in other
SOCS-3-transfected cells (25, 26, 27, 28, 29, 30). In contrast, tyrosine
phosphorylation of STAT3 in response to IFN- was not influenced by
the presence of SOCS-3 (Fig. 8B), excluding that the lack of
responsiveness to IL-10 or IL-6 was due to a general inability to
phosphorylate STAT3 on tyrosine residues by activated JS clones.
Consistent with these results, IL-10- and IL-6-induced DNA-binding
activities to the oligonucleotide probe hSIE/m67 were severely reduced
in JS clones (data not shown).
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production
in JS clones. As shown in Figs. 3B and 4A,
although NO and TNF- production triggered by LPS treatment was
already significantly inhibited by forced SOCS-3 expression, IL-10 was
able to further suppress these functions in JS clones. Treatment of
J774 and JS clones with IL-10 in fact inhibited LPS-induced NO at
24 h by an average of 61 ± 5% in J774 (n =
6; p < 0.001), 36 ± 16% in JS#11
(n = 4; p < 0.05), 81 ± 4% in
JS#13 (n = 2; p < 0.05), and 69
± 16% in JS#L (n = 3; p < 0.05), and
LPS-induced TNF- at 6 h by an average of 80 ± 16% in
J774 (n = 4; p < 0.005), 37 ±
10% in JS#11 (n = 4; p < 0.005), and
67 ± 4% in JS#10 (n = 2; p 
0.01). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our data show that SOCS-3, stably transfected in the J774 cell line at
levels comparable to those of endogenous SOCS-3 induced in response to
IL-10 plus LPS in parental cells, inhibited LPS-induced production of
several proinflammatory mediators, including TNF-, IL-6, GM-CSF, and
NO. SOCS-3 transfection did not, however, influence TNF- or NO
induction stimulated by LPS used in combination with IFN-, thereby
demonstrating that its effects were selective. SOCS-3-mediated
inhibitory effects were quantitatively comparable to the degree of
inhibition exerted by IL-10 on LPS-stimulated wild-type cells and were
achieved by molecular mechanisms acting at both transcriptional and
post-transcriptional levels that resembled those used by IL-10
(10, 11, 31, 32, 33). For instance, we found that while
LPS-induced TNF- production was dramatically inhibited in JS clones
or by IL-10 in J774 cells, inhibition of LPS-induced TNF- mRNA
accumulation was marginal. Although we cannot exclude regulation at the
level of TNF- secretion, our data support the hypothesis recently
proposed by Kontoyiannis and colleagues that IL-10-activated SOCS-3
might block the signaling pathway(s) required to activate
post-transcriptional mechanisms regulating TNF- synthesis
(34). LPS-induced activation of MAPK/SAPK such as ERK,
JNK, and p38, has been shown to regulate TNF- gene expression at the
level of translation (35, 36, 37). In this context, whether
IL-10 exerts its inhibitory action by interfering with the LPS-induced
activation of ERK, JNK, or p38 is not clear, since the results reported
in the literature are contradictory (38, 39, 40, 41, 42). The data
presented in this study, showing that LPS-induced activation of ERK1/2,
SAPK/JNK, and p38 MAPK remain unaltered following IL-10 addition in
wild-type J774, are in line with other work reporting similar
observations (16, 43, 44). Similarly, we show that in JS
clones also the activation of the various MAPK/SAPK signaling pathways
by LPS is unaffected by SOCS-3, adding further evidence that IL-10 and
SOCS-3 actions proceed in parallel.
In contrast with the effects of IL-10 and SOCS-3 on TNF- expression
and production, we consistently observed a correlation between the
decrease in iNOS mRNA levels and the suppression of NO production in
parental J774 cells treated with LPS in the presence of IL-10 as well
as in JS clones after LPS stimulation. The data confirm the inhibitory
effect of SOCS-3 constitutive expression on LPS-dependent NO synthesis
in the murine M1 myeloid cell line previously reported
(13). Moreover, either IL-10 treatment or SOCS-3 forced
expression in LPS-activated J774 caused the inhibition of IL-6 mRNA
expression and protein secretion. This is in contrast with the result
obtained in M1 cell line, in which SOCS-3 forced expression did not
affect LPS-induced expression of IL-6 mRNA (13). However,
we have recently observed that in those M1-trasfected cells,
cytoplasmic myc-SOCS-3 is expressed at lower levels than
those detected in JS clones (C. Berlato, A. Yoshimura,
unpublished observations). We would therefore tend to speculate that in
M1 cells the amount of transfected SOCS-3 is not enough to prevent
LPS-induced IL-6 gene expression.
Another novel and very interesting observation uncovered by our study is that cells expressing SOCS-3, unlike parental J774, constitutively accumulate IL-1ra mRNA transcripts. Addition of LPS to JS clones further enhanced IL-1ra mRNA expression, indicating first that LPS responses are not generally inhibited in JS clones, and secondly that SOCS-3 constitutive expression also reproduces the IL-10-enhancing properties on LPS-mediated induction of the IL-1ra gene (8, 23). Whether SOCS-3 induces expression of IL-1ra mRNA directly via transcription factor activation or through a positive modulation of IL-1ra mRNA stability, similar to the effect of IL-10 in LPS-stimulated neutrophils (23), remains to be established. Nonetheless, in consideration of the anti-inflammatory effects of IL-1ra, these data further support an involvement of SOCS-3 in those mechanisms by which IL-10 exerts its immunosuppressive actions by up-regulating anti-inflammatory molecules.
In addition to the findings that SOCS-3 constitutive expression
inhibits LPS-induced proinflammatory responses in J774 cells, we
demonstrate that SOCS-3 is required for the IL-10-mediated deactivation
of primary peritoneal macrophages treated with LPS. SOCS-3-null
macrophages are not available, since homozygous disruption of the
SOCS-3 gene leads to embryonic lethality as a consequence of
erythrocytosis or abnormal placental development (18, 31).
We therefore took advantage of mice carrying heterozygous
disruption of the SOCS-3 gene, which are phenotypically normal and
fertile (18). We observed that in primary peritoneal
SOCS-3+/- macrophages the induction of SOCS-3
protein expression in response to LPS plus IL-10 was severely impaired.
In parallel with such reduced induction of cytoplasmic SOCS-3, the
inhibitory activity of IL-10 on LPS-induced TNF- and NO production
was significantly reduced, clearly indicating that SOCS-3 is involved
in the negative signaling pathways triggered by IL-10.
Taken together, our observations represent the first evidence that
SOCS-3 induced by IL-10 takes part in the inhibitory pathways activated
by this cytokine and point out for a broad role for SOCS-3 protein in
regulating macrophage deactivation by inhibiting the production of
proinflammatory signals and by favoring the expression of
anti-inflammatory molecules as well. Future work, aimed at
understanding the molecular mechanism(s) by which SOCS-3 mediates
suppression of the LPS signaling pathway, will certainly help in
understanding the intracellular mechanisms by which IL-10 inhibits
proinflammatory cytokine production by LPS-activated phagocytes. In
regard to the latter issue, it has been reported that IL-10
down-regulates the expression of TLR4, the signal transducing receptor
for LPS (32). However, because induction of TNF-,
IL-1ra, and macrophage inflammatory protein-2 (not shown) mRNAs in
response to LPS was not significantly impaired in JS clones compared
with J774 parental cells, we would exclude the possibility that the
impairment of LPS responses observed in SOCS-3-transfected cells was a
consequence of a decreased expression of TLR4.
In this study we also report, for the first time, that STAT3 tyrosine
phosphorylation in response to IL-10 and STAT3 DNA-binding activities
as well are severely impaired in SOCS-3-transfected cells. A critical
role for STAT3 in mediating IL-10-induced inhibition has been
demonstrated by targeted deletion of the STAT3 gene in macrophages and
neutrophils (33). However, it has also been demonstrated
that STAT3 activation is not sufficient to mediate the
anti-inflammatory action of IL-10 (34, 40). In our
experiments we found that SOCS-3 constitutive expression, despite its
inhibitory effect on IL-10-induced STAT3 tyrosine phosphorylation, did
not completely abolish the down-regulatory activities of IL-10 on
LPS-induced NO and TNF- production. Although we cannot exclude that
the minimal tyrosine phosphorylation of STAT3 induced by IL-10 in JS
clones was sufficient to mediate the residual inhibitory effects of
IL-10, as a whole our data support the view that IL-10-mediated
inhibition of TNF- and NO production occurs through STAT3-dependent
and STAT3-independent pathways (21). However, we cannot
rule out the possibility that SOCS-3 forced expression was not
sufficient to completely block the capacity of IL-10 to activate STAT3.
Whatever the case, our data support the view that that an accessory
signaling pathway, in addition to the Jak-STAT pathway, is required for
expression of the anti-inflammatory actions of IL-10
(22).
Defects in IL-10 production or in macrophage STAT3 signaling molecule (5, 33) or forced expression of a dominant negative form of SOCS-3 in mice (30) lead to the development or the worsening of chronic inflammatory pathologies such as inflammatory bowel disease or colitis. A detailed dissection of the mechanisms responsible for SOCS-3 activation and the identification of SOCS-3 intracellular targets is therefore essential for preventing immune dysregulation and chronic inflammatory diseases.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Flavia Bazzoni, Department of Pathology, Section of General Pathology, University of Verona, Strada le Grazie, 37134 Verona, Italy. E-mail address: flavia.bazzoni{at}univr.it
3 Abbreviations used in this paper: SOCS, suppressor of cytokine signaling; ERK1/2, extracellular signal-regulated kinase 1/2; IL-1ra, IL-1R antagonist; iNOS, inducible NO synthase; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; RPA, ribonuclease protection assay; SAPK, stress-activated protein kinase.
Received for publication September 10, 2001. Accepted for publication April 2, 2002.
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 J. Dagvadorj, Y. Naiki, G. Tumurkhuu, F. Hassan, S. Islam, N. Koide, I. Mori, T. Yoshida, and T. Yokochi Interleukin-10 inhibits tumor necrosis factor-{alpha} production in lipopolysaccharide-stimulated RAW 264.7 cells through reduced MyD88 expression Innate Immunity, April 1, 2008; 14(2): 109 - 115. [Abstract] [PDF]
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 M. A. Taubman, X. Han, K. B. LaRosa, S. S. Socransky, and D. J. Smith Periodontal Bacterial DNA Suppresses the Immune Response to Mutans Streptococcal Glucosyltransferase Infect. Immun., August 1, 2007; 75(8): 4088 - 4096. [Abstract] [Full Text] [PDF]
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 S. A. Robertson, A. S. Care, and R. J. Skinner Interleukin 10 Regulates Inflammatory Cytokine Synthesis to Protect Against Lipopolysaccharide-Induced Abortion and Fetal Growth Restriction in Mice Biol Reprod, May 1, 2007; 76(5): 738 - 748. [Abstract] [Full Text] [PDF]
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 M. Wang, T. Markel, P. Crisostomo, C. Herring, K. K. Meldrum, K. D. Lillemoe, and D. R. Meldrum Deficiency of TNFR1 protects myocardium through SOCS3 and IL-6 but not p38 MAPK or IL-1beta Am J Physiol Heart Circ Physiol, April 1, 2007; 292(4): H1694 - H1699. [Abstract] [Full Text] [PDF]
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 T. Chareonsirisuthigul, S. Kalayanarooj, and S. Ubol Dengue virus (DENV) antibody-dependent enhancement of infection upregulates the production of anti-inflammatory cytokines, but suppresses anti-DENV free radical and pro-inflammatory cytokine production, in THP-1 cells J. Gen. Virol., February 1, 2007; 88(2): 365 - 375. [Abstract] [Full Text] [PDF]
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 H. Qin, C. A. Wilson, K. L. Roberts, B. J. Baker, X. Zhao, and E. N. Benveniste IL-10 Inhibits Lipopolysaccharide-Induced CD40 Gene Expression through Induction of Suppressor of Cytokine Signaling-3 J. Immunol., December 1, 2006; 177(11): 7761 - 7771. [Abstract] [Full Text] [PDF]
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V. A. Dennis, A. Jefferson, S. R. Singh, F. Ganapamo, and M. T. Philipp Interleukin-10 Anti-Inflammatory Response to Borrelia burgdorferi, the Agent of Lyme Disease: a Possible Role for Suppressors of Cytokine Signaling 1 and 3. Infect. Immun., October 1, 2006; 74(10): 5780 - 5789. [Abstract] [Full Text] [PDF]
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Y. Li, N. Chu, A. Rostami, and G.-X. Zhang Dendritic Cells Transduced with SOCS-3 Exhibit a Tolerogenic/DC2 Phenotype That Directs Type 2 Th Cell Differentiation In Vitro and In Vivo J. Immunol., August 1, 2006; 177(3): 1679 - 1688. [Abstract] [Full Text] [PDF]
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H. Cheon, Y. H. Rho, S. J. Choi, Y. H. Lee, G. G. Song, J. Sohn, N. H. Won, and J. D. Ji Prostaglandin E2 Augments IL-10 Signaling and Function J. Immunol., July 15, 2006; 177(2): 1092 - 1100. [Abstract] [Full Text] [PDF]
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H. Weighardt, J. Mages, G. Jusek, S. Kaiser-Moore, R. Lang, and B. Holzmann Organ-Specific Role of MyD88 for Gene Regulation during Polymicrobial Peritonitis. Infect. Immun., June 1, 2006; 74(6): 3618 - 3632. [Abstract] [Full Text] [PDF]
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 Z. Chen, A. Laurence, Y. Kanno, M. Pacher-Zavisin, B.-M. Zhu, C. Tato, A. Yoshimura, L. Hennighausen, and J. J. O'Shea Selective regulatory function of Socs3 in the formation of IL-17-secreting T cells PNAS, May 23, 2006; 103(21): 8137 - 8142. [Abstract] [Full Text] [PDF]
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 P. Qasimi, A. Ming-Lum, A. Ghanipour, C. J. Ong, M. E. Cox, J. Ihle, N. Cacalano, A. Yoshimura, and A. L-F. Mui Divergent Mechanisms Utilized by SOCS3 to Mediate Interleukin-10 Inhibition of Tumor Necrosis Factor {alpha} and Nitric Oxide Production by Macrophages J. Biol. Chem., March 10, 2006; 281(10): 6316 - 6324. [Abstract] [Full Text] [PDF]
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A. Ozaki, Y.-i. Seki, A. Fukushima, and M. Kubo The Control of Allergic Conjunctivitis by Suppressor of Cytokine Signaling (SOCS)3 and SOCS5 in a Murine Model J. Immunol., October 15, 2005; 175(8): 5489 - 5497. [Abstract] [Full Text] [PDF]
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S. A. Litherland, K. M. Grebe, N. S. Belkin, E. Paek, J. Elf, M. Atkinson, L. Morel, M. J. Clare-Salzler, and M. McDuffie Nonobese Diabetic Mouse Congenic Analysis Reveals Chromosome 11 Locus Contributing to Diabetes Susceptibility, Macrophage STAT5 Dysfunction, and Granulocyte-Macrophage Colony-Stimulating Factor Overproduction J. Immunol., October 1, 2005; 175(7): 4561 - 4565. [Abstract] [Full Text] [PDF]
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 M. Kamimura, C. Viedt, A. Dalpke, M. E. Rosenfeld, N. Mackman, D. M. Cohen, E. Blessing, M. Preusch, C. M. Weber, J. Kreuzer, et al. Interleukin-10 Suppresses Tissue Factor Expression in Lipopolysaccharide-Stimulated Macrophages via Inhibition of Egr-1 and a Serum Response Element/MEK-ERK1/2 Pathway Circ. Res., August 19, 2005; 97(4): 305 - 313. [Abstract] [Full Text] [PDF]
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A. A. R. Rahimi, K. Gee, S. Mishra, W. Lim, and A. Kumar STAT-1 Mediates the Stimulatory Effect of IL-10 on CD14 Expression in Human Monocytic Cells J. Immunol., June 15, 2005; 174(12): 7823 - 7832. [Abstract] [Full Text] [PDF]
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B. A. Butcher, L. Kim, A. D. Panopoulos, S. S. Watowich, P. J. Murray, and E. Y. Denkers Cutting Edge: IL-10-Independent STAT3 Activation by Toxoplasma gondii Mediates Suppression of IL-12 and TNF-{alpha} in Host Macrophages J. Immunol., March 15, 2005; 174(6): 3148 - 3152. [Abstract] [Full Text] [PDF]
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B. J. Johnson, T. T. T. Le, C. A. Dobbin, T. Banovic, C. B. Howard, F. d. M. L. Flores, D. Vanags, D. J. Naylor, G. R. Hill, and A. Suhrbier Heat Shock Protein 10 Inhibits Lipopolysaccharide-induced Inflammatory Mediator Production J. Biol. Chem., February 11, 2005; 280(6): 4037 - 4047. [Abstract] [Full Text] [PDF]
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 D. Spight, B. Zhao, M. Haas, S. Wert, A. Denenberg, and T. P. Shanley Immunoregulatory effects of regulated, lung-targeted expression of IL-10 in vivo Am J Physiol Lung Cell Mol Physiol, February 1, 2005; 288(2): L251 - L265. [Abstract] [Full Text] [PDF]
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 G. Grutz New insights into the molecular mechanism of interleukin-10-mediated immunosuppression J. Leukoc. Biol., January 1, 2005; 77(1): 3 - 15. [Abstract] [Full Text] [PDF]
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P. O. Anderson, A. Sundstedt, Z. Yazici, S. Minaee, R. Woolf, K. Nicolson, N. Whitley, L. Li, S. Li, D. C. Wraith, et al. IL-2 Overcomes the Unresponsiveness but Fails to Reverse the Regulatory Function of Antigen-Induced T Regulatory Cells J. Immunol., January 1, 2005; 174(1): 310 - 319. [Abstract] [Full Text] [PDF]
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S. E. Erfurth, S. Grobner, U. Kramer, D. S. J. Gunst, I. Soldanova, M. Schaller, I. B. Autenrieth, and S. Borgmann Yersinia enterocolitica Induces Apoptosis and Inhibits Surface Molecule Expression and Cytokine Production in Murine Dendritic Cells Infect. Immun., December 1, 2004; 72(12): 7045 - 7054. [Abstract] [Full Text] [PDF]
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Y. Liu, W. Dong, L. Chen, R. Xiang, H. Xiao, G. De, Z. Wang, and Y. Qi BCL10 Mediates Lipopolysaccharide/Toll-like Receptor-4 Signaling through Interaction with Pellino2 J. Biol. Chem., September 3, 2004; 279(36): 37436 - 37444. [Abstract] [Full Text] [PDF]
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S. Bhattacharyya, P. Sen, M. Wallet, B. Long, A. S. Baldwin Jr, and R. Tisch Immunoregulation of dendritic cells by IL-10 is mediated through suppression of the PI3K/Akt pathway and of I{kappa}B kinase activity Blood, August 15, 2004; 104(4): 1100 - 1109. [Abstract] [Full Text] [PDF]
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L. Li, L. M. Gronning, P. O. Anderson, S. Li, K. Edvardsen, J. Johnston, D. Kioussis, P. R. Shepherd, and P. Wang Insulin Induces SOCS-6 Expression and Its Binding to the p85 Monomer of Phosphoinositide 3-Kinase, Resulting in Improvement in Glucose Metabolism J. Biol. Chem., August 13, 2004; 279(33): 34107 - 34114. [Abstract] [Full Text] [PDF]
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 N. Veal, C.-L. Hsieh, S. Xiong, J. M. Mato, S. Lu, and H. Tsukamoto Inhibition of lipopolysaccharide-stimulated TNF-{alpha} promoter activity by S-adenosylmethionine and 5'-methylthioadenosine Am J Physiol Gastrointest Liver Physiol, August 1, 2004; 287(2): G352 - G362. [Abstract] [Full Text] [PDF]
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 C. Brender, R. Columbus, D. Metcalf, E. Handman, R. Starr, N. Huntington, D. Tarlinton, N. Odum, S. E. Nicholson, N. A. Nicola, et al. SOCS5 Is Expressed in Primary B and T Lymphoid Cells but Is Dispensable for Lymphocyte Production and Function Mol. Cell. Biol., July 1, 2004; 24(13): 6094 - 6103. [Abstract] [Full Text] [PDF]
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H. Gao, R.-F. Guo, C. L. Speyer, J. Reuben, T. A. Neff, L. M. Hoesel, N. C. Riedemann, S. D. McClintock, J. V. Sarma, N. Van Rooijen, et al. Stat3 Activation in Acute Lung Injury J. Immunol., June 15, 2004; 172(12): 7703 - 7712. [Abstract] [Full Text] [PDF]
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P. Perrier, F. O. Martinez, M. Locati, G. Bianchi, M. Nebuloni, G. Vago, F. Bazzoni, S. Sozzani, P. Allavena, and A. Mantovani Distinct Transcriptional Programs Activated by Interleukin-10 with or without Lipopolysaccharide in Dendritic Cells: Induction of the B Cell-Activating Chemokine, CXC Chemokine Ligand 13 J. Immunol., June 1, 2004; 172(11): 7031 - 7042. [Abstract] [Full Text] [PDF]
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L. Zhou, A. A. Nazarian, and S. T. Smale Interleukin-10 Inhibits Interleukin-12 p40 Gene Transcription by Targeting a Late Event in the Activation Pathway Mol. Cell. Biol., March 15, 2004; 24(6): 2385 - 2396. [Abstract] [Full Text] [PDF]
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S. H. Jackson, C.-R. Yu, R. M. Mahdi, S. Ebong, and C. E. Egwuagu Dendritic Cell Maturation Requires STAT1 and Is under Feedback Regulation by Suppressors of Cytokine Signaling J. Immunol., February 15, 2004; 172(4): 2307 - 2315. [Abstract] [Full Text] [PDF]
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T. Katakura, M. Miyazaki, M. Kobayashi, D. N. Herndon, and F. Suzuki CCL17 and IL-10 as Effectors That Enable Alternatively Activated Macrophages to Inhibit the Generation of Classically Activated Macrophages J. Immunol., February 1, 2004; 172(3): 1407 - 1413. [Abstract] [Full Text] [PDF]
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A. C. Morrison, C. B. Wilson, M. Ray, and P. H. Correll Macrophage-Stimulating Protein, the Ligand for the Stem Cell-Derived Tyrosine Kinase/RON Receptor Tyrosine Kinase, Inhibits IL-12 Production by Primary Peritoneal Macrophages Stimulated with IFN-{gamma} and Lipopolysaccharide J. Immunol., February 1, 2004; 172(3): 1825 - 1832. [Abstract] [Full Text] [PDF]
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M. Cvetanovic and D. S. Ucker Innate Immune Discrimination of Apoptotic Cells: Repression of Proinflammatory Macrophage Transcription Is Coupled Directly to Specific Recognition J. Immunol., January 15, 2004; 172(2): 880 - 889. [Abstract] [Full Text] [PDF]
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L. Williams, L. Bradley, A. Smith, and B. Foxwell Signal Transducer and Activator of Transcription 3 Is the Dominant Mediator of the Anti-Inflammatory Effects of IL-10 in Human Macrophages J. Immunol., January 1, 2004; 172(1): 567 - 576. [Abstract] [Full Text] [PDF]
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 I. Lavon, T. Sheinin, S. Meilin, E. Biton, A. Weksler, G. Efroni, A. Bar-Joseph, G. Fink, and A. Avraham A Novel Synthetic Cannabinoid Derivative Inhibits Inflammatory Liver Damage via Negative Cytokine Regulation Mol. Pharmacol., December 1, 2003; 64(6): 1334 - 1341. [Abstract] [Full Text] [PDF]
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H. Kuwata, Y. Watanabe, H. Miyoshi, M. Yamamoto, T. Kaisho, K. Takeda, and S. Akira IL-10-inducible Bcl-3 negatively regulates LPS-induced TNF-{alpha} production in macrophages Blood, December 1, 2003; 102(12): 4123 - 4129. [Abstract] [Full Text] [PDF]
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 A. Kano, M. J. Wolfgang, Q. Gao, J. Jacoby, G.-X. Chai, W. Hansen, Y. Iwamoto, J. S. Pober, R. A. Flavell, and X.-Y. Fu Endothelial Cells Require STAT3 for Protection against Endotoxin-induced Inf lammation J. Exp. Med., November 17, 2003; 198(10): 1517 - 1525. [Abstract] [Full Text] [PDF]
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P. S. Grutkoski, Y. Chen, C. S. Chung, and A. Ayala Sepsis-induced SOCS-3 expression is immunologically restricted to phagocytes J. Leukoc. Biol., November 1, 2003; 74(5): 916 - 922. [Abstract] [Full Text] [PDF]
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A. M. Mackler, T. C. Ducsay, C. A. Ducsay, and S. M. Yellon Effects of Endotoxin and Macrophage-Related Cytokines on the Contractile Activity of the Gravid Murine Uterus Biol Reprod, October 1, 2003; 69(4): 1165 - 1169. [Abstract] [Full Text] [PDF]
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R. Biswas, S. Datta, J. D. Gupta, M. Novotny, J. Tebo, and T. A. Hamilton Regulation of Chemokine mRNA Stability by Lipopolysaccharide and IL-10 J. Immunol., June 15, 2003; 170(12): 6202 - 6208. [Abstract] [Full Text] [PDF]
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 K. Asadullah, W. Sterry, and H. D. Volk Interleukin-10 Therapy--Review of a New Approach Pharmacol. Rev., June 1, 2003; 55(2): 241 - 269. [Abstract] [Full Text] [PDF]
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M. Schroder, C. Meisel, K. Buhl, N. Profanter, N. Sievert, H.-D. Volk, and G. Grutz Different Modes of IL-10 and TGF-{beta} to Inhibit Cytokine-Dependent IFN-{gamma} Production: Consequences for Reversal of Lipopolysaccharide Desensitization J. Immunol., May 15, 2003; 170(10): 5260 - 5267. [Abstract] [Full Text] [PDF]
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Y. Ding, D. Chen, A. Tarcsafalvi, R. Su, L. Qin, and J. S. Bromberg Suppressor of Cytokine Signaling 1 Inhibits IL-10-Mediated Immune Responses J. Immunol., February 1, 2003; 170(3): 1383 - 1391. [Abstract] [Full Text] [PDF]
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X. Li, P. E. Massa, A. Hanidu, G. W. Peet, P. Aro, A. Savitt, S. Mische, J. Li, and K. B. Marcu IKKalpha , IKKbeta , and NEMO/IKKgamma Are Each Required for the NF-kappa B-mediated Inflammatory Response Program J. Biol. Chem., November 15, 2002; 277(47): 45129 - 45140. [Abstract] [Full Text] [PDF]
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 M. J. Scott, C. J. Godshall, and W. G. Cheadle Jaks, STATs, Cytokines, and Sepsis Clin. Vaccine Immunol., November 1, 2002; 9(6): 1153 - 1159. [Full Text] [PDF]
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) or
constitutive SOCS-3 expression in JS#11 (
) and JS#13 (
). Values
are the mean ± SE of four independent experiments for protein
secretion and three experiments for mRNA expression.
