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*
Department of Medicine, Hospital for Special Surgery, and
Graduate Program in Immunology, Weill Graduate School of Medical Sciences, Cornell University, New York, NY 10021
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, are expressed in the majority
of patients with RA. The known activity of proinflammatory cytokines
detected in inflamed synovium can account for many processes important
in joint destruction: production of proteases and reactive oxygen
intermediates, synovial fibroblast (SF) proliferation, cartilage
degradation, influx of inflammatory cells, and angiogenesis. The most
compelling support for cytokines in RA pathogenesis comes from the
attenuation of arthritis by therapies aimed at modulating or blocking
cytokine activity (2, 5). Exciting studies have
demonstrated dramatic improvement in synovial inflammation in RA
patients after treatment with neutralizing anti-TNF- Abs or
soluble TNF receptors (5, 6, 7), and decreased joint
destruction after treatment with IL-1R antagonist (IL-1RA) (Ref.
8 and references therein). Interestingly,
immunosuppressive and anti-inflammatory cytokines, including
TGF
, IL-10, and IL-1RA, are highly and consistently expressed during
RA synovitis (1, 2). Production of these cytokines has
been proposed to reflect the patients’ attempts to contain or control
inflammation and achieve homeostasis (3, 4). Treatment
with TGF, IL-10, and IL-1RA has been successful in ameliorating
disease in acute animal models of arthritis, such as collagen-induced
arthritis (CIA). An interesting question is why the high endogenous
levels of anti-inflammatory factors that are expressed in RA joints
are unable to suppress long-term chronic synovitis in patients.
IL-6 is one member of a family of related cytokines (IL-6,
IL-11, oncostatin M (OsM), LIF, and cardiotropin) that share the gp130
signaling receptor subunit. IL-6, IL-11, OsM, and LIF are highly
expressed during RA synovitis (1, 9). Although these
cytokines are pleiotropic and can promote immune responses, they also
have clear-cut anti-inflammatory effects, especially on macrophages
and fibroblasts. For example, IL-11 suppresses expression of IL-12,
IFN-
, TNF-, adhesion molecules, and proteases
(10, 11, 12, 13), inhibits cytokine production in RA synovium
(9), and has been used successfully to treat CIA
(14) and psoriasis (15). In contrast to
IL-11, IL-6 appears to have different actions on different cell types
and can act to either promote or suppress inflammatory arthritis.
Consistent with its known stimulatory effects on T and B cells, IL-6
appears to be critically important in the initiation phase of CIA,
which is mediated by lymphocyte responses against type II collagen
(16, 17). In addition, IL-6 induces chemokine expression
in endothelial cells (18). In contrast to its effects on
lymphocytes and endothelial cells, IL-6 has been reported to have
suppressive effects on other cell types, including macrophages and SFs,
that are important in RA pathogenesis. The reported
anti-inflammatory effects of IL-6 include induction of
anti-inflammatory cytokines such as IL-1RA, induction of acute
phase reactants that subserve anti-inflammatory functions,
induction of glucocorticoid production, suppression of cytokine
production (IL-1, TNF, and IL-12) and adhesion molecule expression,
suppression of proliferation of RA SFs, inhibition of protease
expression, and induction of protease inhibitors (such as TIMP-1) in RA
SFs (19, 20, 21, 22, 23, 24, 25, 26, 27, 28). IL-6 induction of TIMP expression in RA SFs
blocks IL-1-induced collagenolytic activity and likely plays an
important role in suppressing the ability of these cells to invade and
destroy cartilage (28). Consistent with these
anti-inflammatory effects, IL-6 ameliorates inflammation in several
animal models, including inflammatory lung disease (29),
and plays a chondroprotective role in zymosan-induced arthritis
(30). The strongest evidence of an anti-inflammatory
role for IL-6 in arthritis is the development of spontaneous arthritis
in mice containing a mutation that partially abrogates IL-6 signal
transduction (31). These results suggest that the role of
IL-6 in RA pathogenesis will depend upon the balance between its pro-
and anti-inflammatory actions on different cell types.
The idea that the balance between pro- and anti-inflammatory
factors is important in regulating the rate of progression, and thus
the eventual severity and morbidity, of RA has gained acceptance among
RA researchers and formed one rationale for developing anti-TNF-
therapies (2). The current concept is that one important
determinant of the balance between pro- and anti-inflammatory
factors is the relative level of expression of, on the one hand,
inflammatory cytokines, and, on the other hand, anti-inflammatory
cytokines, receptor antagonists, and soluble receptors (2, 3). However, it is becoming increasingly clear that cytokine
effects can be blocked intracellularly at the level of signal
transduction. For example, the potent anti-inflammatory cytokine
IL-10 blocks signaling by IFN- (32), and IL-6 and IL-11
block signaling and NF-
B activation by LPS (33).
Conversely, IFN- and IL-1 block signaling by TGF (34, 35). Thus, the final effect of cytokines on cellular phenotype
is not determined solely by the relative levels of expression of
different cytokines. We hypothesized that one mechanism by which
inflammatory cytokines perpetuate synovitis in the presence of
anti-inflammatory factors in the synovium is through inhibition or
modulation of signaling by anti-inflammatory or pleiotropic
cytokines. This hypothesis was tested by analyzing the effects of IL-1
and TNF on IL-6 signaling in RA SFs, cells in which IL-6 has effects
consistent with an anti-inflammatory role, such as inhibition of
proliferation and induction of TIMP-1 production (27, 28).
Our results show that Janus kinase (Jak)-STAT signaling by IL-6 in RA
synoviocytes is inhibited by IL-1 and TNF-. These results identify a
novel level of cytokine cross-talk in RA synovium and suggest that
cytokine antagonism at the level of signal transduction contributes to
the balance of cytokine activity, and thus pathogenesis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Synovial tissues were obtained perioperatively from patients who fulfilled the revised American College of Rheumatology criteria for definite RA and were undergoing total joint replacement; the protocol was approved by the Institutional Review Board of the Hospital for Special Surgery (New York, NY). A total of 24 specimens from different donors were analyzed. Synovial cells were obtained by finely mincing freshly isolated synovial tissue, followed by treatment with collagenase A (1 mg/ml; Boehringer Mannheim, Indianapolis, IN) for 2 h at 37°C. Fibroblasts were obtained by allowing cells to adhere to tissue culture plates, followed by removal of nonadherent cells. The initially plated adherent cells contained contaminating macrophages, and, in experiments in which cells were used before in vitro culture and fibroblast expansion, macrophages were removed using anti-CD14 magnetic beads (Miltenyi Biotec, Auburn, CA), as previously described (36). Similar to previous reports, most of the experiments were performed using SFs between the third and fifth passages in tissue culture. At the third passage, there were <2% contaminating lymphocytes, NK cells, or macrophages, as assessed by flow cytometry and staining with Abs against CD3, CD14, CD16, and CD19, as previously described (36). SFs were cultured in DMEM supplemented with 10% FBS, and cells were routinely split and replated the day before an experiment.
EMSAs
Cell extracts were prepared as previously described (37) and protein concentration was determined using the Bradford assay (Bio-Rad, Hercules, CA). Equal amounts of protein (typically 10 µg) were incubated for 15 min at room temperature with 0.5 ng of 32P-labeled double-stranded hSIE oligonucleotide (38) in a 15-µl binding reaction containing 40 mM NaCl and 2 µg of poly(dI-dC) (Pharmacia, Piscataway, NJ), as previously described (37), and complexes were resolved on nondenaturing 4.5% polyacrylamide gels.
Immunoblotting
Cell lysates were fractionated on 7.5% SDS-polyacrylamide gels, transferred to polyvinylidene difluoride membranes, and incubated with phosphospecific (Tyr705) Stat3 Ab, phosphospecific (Thr180/Tyr182) p38 Ab, phosphospecific (Thr202/Tyr204) extracellular stimulus-regulated kinase (ERK)1/2 Ab (New England Biolabs, Beverly, MA), monoclonal Stat3 and ERK1/2 Abs (BD Transduction Laboratories, Lexington, KY), and p38 Abs (Santa Cruz Biotechnology, Santa Cruz, CA). ECL was used for detection.
ELISA
Paired TIMP-1 capture and detection Abs and TIMP-1 protein were purchased and used in a sandwich ELISA according to the instructions of the manufacturer (R&D Systems, Minneapolis, MN).
Proliferation analysis
Cells (104) in 100 µl of medium were seeded in triplicate in 96-well tissue culture plates, cytokines were added after 8 h, and cells were cultured for an additional 24 h. Cells were pulsed for the final 8 h of culture with 10 µCi/ml [3H]thymidine and harvested using an automated cell harvester (Harvester 96; Tomtec, Orange, CT), and [3H]thymidine incorporation was quantitated using a Wallac Microbeta Trilux Scintillation Counter (PerkinElmer Wallac, Gaithersburg, MD). Cell counts were performed on parallel wells in duplicate and cell viability was determined using trypan blue and propidium iodide exclusion.
Analysis of mRNA levels
For semiquantitative RT-PCR, total cellular RNA was isolated
using TRIzol (Life Technologies, Gaithersburg, MD) according to the
instructions of the manufacturer. RNA was treated with RNase-free
DNase, and cDNA was obtained using Moloney murine leukemia virus
reverse transcriptase (Life Technologies). A total of 2.5% of each
cDNA was subjected to 22–25 cycles of PCR using conditions that result
in a single specific amplification product of the correct size, as
previously described (36, 39): 30 s denaturation at
94°C, 1 min annealing at 55°C, and 30 s extension at 72°C in
a GeneAmp 9600 thermal cycler (PerkinElmer, Norwalk, CT). dNTPs were
used at 100 µM and 1 µCi of [32P]-dATP
was added to each reaction. No amplification products were obtained
when reverse transcriptase was omitted, indicating the absence of
contaminating genomic DNA. Amplification was empirically determined to
be in the linear range. For real-time, quantitative PCR, DNA-free RNA
was obtained using the RNeasy Mini kit from Qiagen (Valencia, CA) with
DNase treatment, and 1 µg of total RNA was reverse-transcribed using
random hexamers and Moloney murine leukemia virus reverse
transcriptase. Real-time PCR was performed in triplicate using the
iCycler iQ thermal cycler and detection system (Bio-Rad) and the PCR
Core Reagents kit (Applied Biosystems, Foster City, CA) with 500-nM
primers. The final Mg2+ concentration was
adjusted to 4 mM. Four-fold serial dilutions of cDNAs were used to
generate curves of log input amount vs threshold cycle, and comparable
slopes for a given primer set were obtained for the group of cDNAs
being tested (signifying comparable efficiencies of amplification).
Fold induction was normalized for levels of GAPDH. When reverse
transcriptase was omitted, threshold cycle number increased by
at least 10, signifying lack of genomic DNA contamination or
nonspecific amplification, and the generation of only the correct size
amplification products was confirmed using agarose gel electrophoresis.
The primer sequences used were: GAPDH, 5'-CGA CGC CTG CTT CAC CAC
CTT-3' and 5'-CGG GGC TCT CCA GAA CAT CAT CC-3'; IFN regulatory factor
(IRF-1), 5'-CTG GCT CCT TTT CCC CTG CTT TGT-3' and 5'-CAA ATC
CCG GGG CTC ATC TGG-3'; suppressor of cytokine signaling
(SOCS)3, 5'-TCG CCC CCG GAG TAG ATG TAA TAG-3' and 5'-CAC TAC
ATG CCG CCC CCT GGA G-3'; IP-10 5'-TCT CAC CCT TCT TTT TCA TTG TAG-3'
and 5'-ATT TGC TGC CTT ATC TTT CTG-3'; monokine induced by IFN-
(MIG), 5'-GCT TTT TCT TTT GGC TGA CCT GTT-3' and 5'-ATC AGC ACC
AAC CAA GGG ACT ATC-3'.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Activation of the Jak-STAT pathway by IL-6 in RA SFs was
investigated by using EMSAs to detect the activation of DNA binding of
STAT transcription factors. Addition of IL-6 alone did not result in
any detectable binding of protein complexes to a hSIE oligonucleotide
that binds Stat1, Stat3, and Stat4 (Fig. 1
A, lane 2);
binding activity was not detected using the IRF oligonucleotide that
binds Stat5 and Stat6 (data not shown). This result is consistent with
previous reports suggesting that SFs express only the gp130 signaling
subunit of the IL-6R, but not the IL-6-binding subunit (27, 28). As previously described in other cells types
(40), addition of extracellular soluble IL-6R subunit
(sIL-6R) together with IL-6 resulted in initiation of signal
transduction, as determined by activation of STAT DNA-binding activity
(Fig. 1A, lane 4). Use of sIL-6R has clinical
relevance, because this factor is elevated in RA and expressed in RA
synovium at concentrations as high as 200 ng/ml (1). DNA
binding was specific as determined by competition experiments with
unlabeled oligonucleotide (data not shown). When SFs were pretreated
with the glucocorticoid dexamethasone (dex), addition of IL-6 alone
activated STAT DNA binding (Fig. 1B), consistent with
previous results that cell surface expression of IL-6R is induced by
glucocorticoids (41). The induction of cellular
responsiveness to IL-6 by the potent immunosuppressive agent dex is
consistent with an anti-inflammatory action of IL-6 on SFs. IL-6
has been reported to activate Stat1 and Stat3 in other cell types, and
supershift experiments with specific Abs confirmed that IL-6 plus
sIL-6R activated both Stat1 and Stat3 in SFs (Fig. 1C).
Although the relative ratio of Stat1 and Stat3 varied among independent
experiments using different donors, activation of these STATs was
consistently detected in SFs that were derived from 24 different
patients and used immediately (after depletion of macrophages and
lymphocytes) or after three, six, or 10 passages. The effect of
addition of the IL-6-related cytokines LIF, OsM, and IL-11 on STAT DNA
binding activity was determined. LIF and OsM activated STAT DNA binding
(Fig. 1D), but IL-11 did not (data not shown). These results
are consistent with previous observations that SFs express the LIF- and
OsM-specific receptor subunits, and respond to IL-11 only if
soluble IL-11R is provided (9). These results
demonstrate that IL-6 and related cytokines activate Stat1 and Stat3 in
RA SFs.
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stimulation on gene expression in SFs.
Third-passage RA SFs were stimulated for 3 h with IL-6 plus
sIL-6R, and mRNA levels for several genes were determined (Fig. 2A). Interestingly, IL-6
induced expression both of genes that may be considered
anti-inflammatory (protein inhibitor of activated Stat1, an
inhibitor of IFN--Stat1 signaling, and TGF), genes that are
considered proinflammatory (complement component 3, and IP-10 (a
chemokine)), and genes whose functions in synoviocytes are not known
(SOCS3 and IRF-1, a transcription factor). This pattern is consistent
with the pleiotropic nature of IL-6, as discussed above. The extent of
gene activation (fold induction) varied among different patients, but a
similar pattern of activation was detected in eight independent
experiments; similar results were obtained when OsM was used (data not
shown). SOCS3 and IRF-1 genes are directly transcriptionally activated
by STATs, and thus these results demonstrate that IL-6 activation of
the Jak-STAT pathway in RA SFs is coupled to transcriptional responses.
Addition of IL-6 and sIL-6R suppressed synoviocyte proliferation,
reproducing recently reported results (27) (Fig. 2B). The level of suppression of proliferation was greater
than that observed with IL-10, which has been shown to be effective in
suppressing RA synoviocytes that have been implanted into SCID mice
(42, 43). Suppression of synoviocyte proliferation (and
induction of TIMP-1 expression; see Fig. 4B) by IL-6 further
demonstrates a functional cellular response to this cytokine and is
consistent with a down-regulatory effect of IL-6 on SFs.
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IL-1 is a potent proinflammatory cytokine expressed in most
inflamed RA joints, and the effect of IL-1 on IL-6 signaling was
determined. IL-1 pretreatment for 20 min strongly blocked activation of
STAT DNA binding, and inhibition was persistent, in that STAT
activation was blocked when the period of incubation with IL-1 was
extended to 3 h (Fig. 3A,
top panel). One representative experiment of over 15
experiments using different RA synovial tissues is shown. Activation of
STAT DNA binding activity is dependent upon tyrosine phosphorylation
(44), and preincubation with IL-1 effectively blocked
Stat3 tyrosine phosphorylation (Fig. 3A, middle
panel) in parallel with inhibition of DNA binding. Stat3 levels
did not change after IL-1 treatment (Fig. 3A, bottom
panel), indicating that the decrease in Stat3 DNA binding was not
secondary to decreased protein levels. A rapid block in STAT DNA
binding and tyrosine phosphorylation suggests that inhibition may occur
at a proximal step in signal transduction, before phosphorylation and
activation.
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B); in
some experiments >50% inhibition of IL-6 signaling was observed using
doses of IL-1 as low as 100 pg/ml (data not shown). Similar to
inhibition of IL-6, IL-1 treatment inhibited activation of STATs by OsM
and LIF (Fig. 3C and data not shown), indicating that IL-1
blocks signaling by members of the IL-6 family of cytokines that share
similar receptors (40) and have anti-inflammatory
effects on synovitis. In contrast, IL-1 had only a weak and partial
inhibitory effect on activation of Stat1 by IFN- (Fig. 3C, lanes 4 and 5); in two additional
experiments no effect of IL-1 on IFN- signaling was detected (data
not shown). IL-1 did not inhibit activation of Stat6 by IL-13 to any
appreciable extent (Fig. 3C). These results demonstrate that
Jak-STAT signaling was not globally blocked by IL-1. Treatment of SFs
with TNF-, which activates similar signaling pathways to those
activated by IL-1, also inhibited IL-6-induced STAT activation (Fig. 3D). These results, taken together, indicate that
proinflammatory cytokines present during RA synovitis inhibit Jak-STAT
signaling by pleiotropic cytokines of the IL-6 family more strongly
than signaling by inflammatory cytokines such as IFN-.
Both synergistic and antagonistic interactions of IL-1 and TNF with
cytokines of the IL-6 family have been described. IL-1 and IL-6 work
together in the induction of type I acute phase protein genes
(21), metalloproteases (45), and HIV
expression (46). In contrast, IL-1 and TNF can block other
IL-6 responses, such as induction of type II acute phase response genes
thiostatin and fibrinogen (21, 47) and IL-6-induced
proliferation of thymocytes (48). Our results suggested
that IL-1 may also block IL-6 responses in RA SFs, especially those
that are dependent on the Jak-STAT signaling pathway. The consequences
of IL-1 pretreatment on IL-6 induction of gene expression were
investigated. Surprisingly, genes that were induced by IL-6 were also
induced by IL-1, and analysis of the interaction of IL-1 and IL-6 was
difficult secondary to the limitations of the semiquantitative nature
of the RT-PCR assay used. Therefore, real-time quantitative PCR was
used to accurately measure expression levels of four genes, IRF-1,
SOCS3, IP-10, and MIG (Fig. 4A; one representative
experiment of four performed is shown). IRF-1 mRNA levels were strongly
induced by IL-6 and, to a lesser extent, by IL-1. Interestingly,
preincubation with IL-1 effectively blocked any additional induction of
IRF-1 expression by IL-6 (Fig. 4A). Because induction of
IRF-1 by type I cytokines and IFNs is entirely dependent upon the
Jak-STAT signaling pathway, this result demonstrates that blocking IL-6
signaling via STATs results in an effective block of IL-6 activation of
a STAT-dependent gene. Although IL-1 itself activated IRF-1 expression,
it also limited the levels to which IRF-1 expression was induced, and
the potential physiological significance of this pattern of regulation
is addressed in Discussion. IL-1 induced expression of SOCS3
mRNA, consistent with previous reports (49, 50, 51), and also
blunted the induction of SOCS3 expression by IL-6 (the effects of IL-6
and IL-1 are clearly not additive, Fig. 4A). In contrast to
IRF-1 and SOCS3, IL-1 and IL-6 synergized in the activation of the
IP-10 and MIG genes (Fig. 4A; this effect was reproducible
in four independent experiments, and a representative experiment is
shown). These results demonstrate a functional correlate to inhibition
of IL-6 activation of STATs and suggest that IL-1 modulates IL-6 action
in a complex fashion.
Modest alterations in early cellular responses are often amplified in
ensuing cascades of signal transduction and expression of regulatory
molecules and transcription factors. Therefore, we tested whether IL-1
may have a more pronounced effect on IL-6 induction of TIMP-1, an
effect that is not observed until 
4 days after addition of IL-6 and
sIL-6R (28). Basal levels of TIMP-1 production varied
among different synovial specimens and, consistent with a previous
report (28), addition of IL-6 plus sIL-6R resulted in
induction of TIMP-1 expression that was detected after 96 h (Fig. 4B). Induction of TIMP-1 expression by IL-6 plus sIL-6R was
effectively blocked by the addition of IL-1 (Fig. 4B). Fig. 4B shows results obtained using SFs from three different RA
patients; an additional three experiments yielded similar results.
Although it is unlikely that Stat3 directly regulates the TIMP-1
promoter, these results show the following: 1) IL-1 can effectively
inhibit IL-6 effects upon RA SF phenotype; 2) IL-1 suppresses IL-6
induction of a molecule, TIMP-1, that blocks IL-1-induced
collagenolytic activity; thus, IL-1 prevents IL-6-mediated feedback
inhibition of IL-1-induced collagenolytic activity; and 3) IL-1 blocks
an IL-6 effect on RA SFs that is clearly anti-inflammatory in terms
of suppressing tissue destruction.
IL-6 signaling is blocked by a mechanism that does not require new RNA synthesis, is not dependent on a tyrosine phosphatase, but is dependent on MAPKs
One widely studied mechanism of inhibition of Jak-STAT signaling
is mediated through inhibitory proteins termed SOCS, JAB, SSI, or CIS
proteins (referred to herein as SOCS proteins) that likely act, at
least in part, by binding to and inactivating Jaks
(52, 53, 54, 55). Inhibition by SOCS proteins depends upon de novo
RNA and protein synthesis, and thus is unlikely to explain inhibition
of IL-6 signaling observed at early time points, such as 20 min after
addition of IL-1, but could potentially contribute to inhibition
observed at later time points (Fig. 3A). The dependence of
IL-1-mediated inhibition on de novo RNA and protein synthesis was
examined. Inhibition of protein synthesis by cycloheximide resulted in
a rapid loss of IL-6 signaling (within 1 h), possibly explained by
loss of IL-6R expression, as previously reported by others
(56). The effect of actinomycin D, an inhibitor of RNA
synthesis and de novo gene expression, was determined. As predicted,
actinomycin D treatment did not affect IL-1 inhibition of IL-6
signaling when IL-1 was added 20 min before adding IL-6 (Fig. 5A, lanes 2 and
3). Surprisingly, blocking de novo RNA synthesis using
actinomycin D did not affect IL-1 inhibition of IL-6-induced STAT
activation when cells were preincubated with IL-1 for longer periods,
including 3 h (Fig. 5A, lanes 4–9). The
efficacy of actinomycin D in blocking RNA synthesis was confirmed in
duplicate wells to those used to make cell extracts (Fig. 5B). Because IL-1 induction of SOCS3 expression may
contribute to inhibition of IL-6 signaling, we were interested in
assessing the effects of actinomycin D treatment on SOCS3 mRNA levels.
Actinomycin D not only completely blocked induction of SOCS3 mRNA
levels by IL-1, but, consistent with previous reports showing that
SOCS3 mRNA is highly unstable, SOCS3 mRNA levels decayed rapidly in
actinomycin D-treated cells, even when IL-1 was added (Fig. 5C); similar results were obtained in the absence of IL-1
(data not shown). Although SOCS3 may regulate IL-6 signaling in the
basal state and may contribute to IL-1-mediated inhibition of IL-6
signaling, this result suggests that IL-1 also triggers an inhibitory
pathway that is independent of SOCS3.
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, lanes 2 and
3). IL-1 effectively blocked IL-6-induced STAT activation
even in the presence of vanadate (Fig. 6, lanes 3–5). These
results indicate that inhibition of IL-6 signaling by IL-1 is not
mediated by a tyrosine phosphatase.
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). The time course of p38 and ERK
activation was typical in that peak levels of activated kinases were
detected 5–15 min after addition of IL-1, and decayed to near baseline
over the next 3 h.
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). Inhibition of p38 resulted in near
complete reversal of IL-1-mediated inhibition (Fig. 8A, lanes 3 and
4), whereas inhibition of ERKs had only a small effect (Fig. 8A, lane 5). Essentially complete inhibition of
ERK activation by PD98059 was verified in the same experiment (data not
shown). These results suggest that rapid inhibition of IL-6 signaling
by IL-1 is mediated by MAPKs that are active when IL-6 is added to
cells, and that p38 plays a more important role than the ERKs in RA
SFs. The effects of inhibiting p38 and ERKs on IL-6 signaling were also
determined when IL-1 was added 3 h before addition of IL-6; at
this time point, p38 and ERK activity had decayed close to baseline
(see Fig. 7). Interestingly, inhibition of p38 resulted in complete
reversal of the IL-1 effect, whereas inhibition of ERKs had a minimal
effect (Fig. 8B). SB203580 has been shown to partially
suppress induction of SOCS3 expression by TNF or CpG-DNA (51, 60). Therefore, these experiments were also conducted in the
presence of actinomycin D, and similar results were obtained (Fig. 8C), suggesting that reversal of inhibition by SB203580 is
not mediated by an effect on SOCS3 expression. Taken together, these
results indicate that MAPKs, predominantly p38, play an important role
in IL-1 inhibition of IL-6 signaling at both early and late time points
after addition of IL-1, and that induction of gene expression and de
novo RNA synthesis are not required for inhibition of IL-6 activation
of STATs.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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are expressed in RA
joints at levels comparable to those used herein, and the p38 kinase is
active during RA synovitis and has been proposed to represent a good
therapeutic target. These results advance the concept of cytokine
balance in RA to the level of signal transduction and suggest that
expression levels of cytokines are not the only factor that regulates
cytokine balance during RA. Manipulation of cytokine signaling to block
the proinflammatory actions of pleiotropic cytokines or to potentiate
the actions of anti-inflammatory cytokines that are already present
in RA synovium may represent a promising novel therapeutic approach
to RA.
Our studies focused upon IL-6, a pleiotropic cytokine that has pro- and
anti-inflammatory effects on different cell types, but overall
likely plays a suppressive role vis a vis SFs (27, 28, 31). These studies could not be extended to the
anti-inflammatory cytokine IL-10, as activation of Jak-STAT
signaling by IL-10 in SFs was not detected (D. Deon, unpublished
results). IL-6 expression is induced by IL-1 and these two cytokines
are coexpressed at many sites of inflammation. Our analysis of the
effects of IL-1 on IL-6 action and IL-6-induced gene expression in RA
synoviocytes revealed that these cytokines interact in a complex
fashion. Several genes that were induced by IL-6 were also induced by
IL-1, and, in the case of IRF-1 and SOCS3, IL-1 suppressed the further
induction of gene expression by IL-6 (Fig. 4A). These
results demonstrate that a block in signal transduction was effectively
translated into a suppression of gene activation. Because IRF-1 and
SOCS3 genes are regulated by STATs, our results identify inhibition of
STAT activation by IL-1 as one molecular mechanism underlying
suppression of IL-6-induced gene expression by IL-1. The pattern of
regulation of IRF-1 and SOCS3 gene expression suggests a negative
feedback loop, whereby IL-1 activates expression of IL-6 and at the
same time limits the ability of IL-6 to further increase expression
levels of these genes. Because IRF-1 and SOCS3 regulate cellular
responses to cytokines and IFNs, we propose that tight regulation of
these genes is important for RA synoviocyte physiology, but the exact
role of these genes in RA inflammation remains to be elucidated.
IL-6 is a pleiotropic cytokine that activates expression of both pro- and anti-inflammatory genes. One may propose two mechanisms by which IL-1 modulation of IL-6 activity may contribute to RA pathogenesis: 1) IL-1-mediated suppression of the IL-6 induction of anti-inflammatory genes; and 2) lack of suppression or superinduction of IL-6 proinflammatory genes by IL-1. Our results identify an example of both of these mechanisms, namely suppression of IL-6 induction of TIMP-1 expression (thus suppressing the ability of IL-6 to limit tissue damage by metalloproteases), and synergistic activation of IP-10 and MIG expression (thus potentiating recruitment of inflammatory cells). These results suggest that IL-1 modulation of IL-6 signaling may contribute to RA pathogenesis.
One interesting aspect of our results is that inhibition of IL-6 STAT
activation by IL-1 had opposite effects on IL-6 induction of different
genes. These results are consistent with previous reports demonstrating
a complex interplay between IL-1 and IL-6, including mutual antagonism
as well as synergy, even in the same cell type (11, 13, 15, 19, 21, 25, 45, 46, 47, 48). For example, IL-1 and IL-6 coactivate
expression of type I acute phase response genes, but IL-1 blocks IL-6
induction of type II acute phase response genes (thiostatin,
fibrinogen) in hepatocytes (21). Divergent effects of
inhibition of STATs on expression of different genes is consistent with
data that suggest that STATs can function as either activators
or inhibitors of transcription of different genes (61, 62). An additional possible molecular basis for divergent
patterns of gene expression is that IL-6 activates signaling pathways
in addition to the Jak-STAT pathway (40) that may work
together with IL-1 signaling pathways. These possibilities are detailed
in Fig. 9. It would be of interest to
test whether p38 plays a role in mediating the effects of IL-1 on
expression of IL-6-inducible genes, similar to its role in modulation
of IL-6 signaling. However, SB203580 alone blocked the induction of
gene expression by IL-6, thus precluding this analysis (N. Scaletta,
unpublished data). These results are in accord with previous
descriptions of a block of Jak-STAT-inducible transcription by
inhibitors of p38, which may work by disrupting interactions of STATs
with transcriptional coactivators, by acting directly on STATs
themselves, or by acting on as yet unknown proteins (63, 64). A more complete understanding of the consequences of IL-1
and IL-6 signal transduction cross-talk on RA synoviocyte phenotype
will require delineation of the roles of Stat1 and Stat3 in RA
synoviocytes, and these experiments are in progress. In contrast to
synoviocytes, IL-1 and TNF did not affect IL-6 signaling in B or T
cells (S. Ahmed, unpublished data), and therefore would not suppress
the proimmune functions of IL-6. Thus, the complex interplay between
IL-1 and IL-6 signaling also varies according to cell type, which has
important implications for the ability of these two cytokines to act
together, or in opposition, in an inflammatory process.
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Acknowledgments |
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Footnotes |
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2 Current address: Hanyang University, Seoul, Korea.
3 Address correspondence and reprint requests to Dr. Lionel B. Ivashkiv, Hospital for Special Surgery, 535 East 70th Street, New York, NY 10021. E-mail address: IvashkivL{at}HSS.edu
4 Abbreviations used in this paper: RA, rheumatoid arthritis; OsM, oncostatin M; sIL-6R, soluble IL-6R subunit; CIA, collagen-induced arthritis; SF, synovial fibroblast; SOCS, suppressor of cytokine signaling; MAPK, mitogen-activated protein kinase; ERK, extracellular stimulus-regulated kinase; Jak, Janus kinase; IL-1RA, IL-1R antagonist; IRF, IFN regulatory factor; dex, dexamethasone; MIG, monokine induced by IFN-.
Received for publication December 6, 2000. Accepted for publication August 23, 2001.
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References |
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