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B But Not C/EBPß or c-Jun1
*
Division of Rheumatology, Department of Medicine and the
Department of Microbiology and Immunology, Northwestern University VA Chicago, Lakeside Medical School, Chicago, IL 60611; and
Division of Biology, California Institute of Technology, Pasadena, CA 91125
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B, C/EBPß, and c-Jun, but the contribution of each to the
regulation of IL-6 and IL-8 in RA FLS is unknown. We employed
adenoviral-mediated gene delivery of a nondegradable IB
, or
dominant-negative versions of C/EBPß or c-Jun, to determine the
contribution of each transcription factor to IL-6 and IL-8 expression.
Inhibition of NF-B activation significantly reduced the spontaneous
and IL-1ß-induced secretion of IL-6 and IL-8 by RA FLS and the
IL-1ß-induced production of IL-6 and IL-8 by human dermal
fibroblasts. Inhibition of C/EBPß modestly reduced constitutive
and IL-1ß-induced IL-6 by RA FLS, but not by human dermal
fibroblasts, and had no effect on IL-8. Inhibition of c-Jun/AP-1 had no
effect on the production of either IL-6 or IL-8. Employing gel shift
assays, NF-B, C/EBPß, and c-Jun were constitutively activated in
RA FLS, but only NF-B and c-Jun activity increased after IL-1ß.
The reduction of cytokines by IB was mediated through inhibition
of NF-B activation, which resulted in decreased IL-6 and IL-8 mRNA.
NF-B was essential for IL-6 expression, because fibroblasts in which
both NF-B p50/p65 genes were deleted failed to express IL-6 in
response to IL-1. These findings document the importance of NF-B for
the regulation of the constitutive and IL-1ß-stimulated expression of
IL-6 and IL-8 by RA FLS and support the role of inhibition of NF-B
as a therapeutic goal in RA. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The transcription factors C/EBPß, AP-1, and NF-B are
constitutively activated in RA synovial tissue (10, 11, 12, 13, 14, 15, 16).
Binding sites for each of these transcription factors have been
identified in the promoter regions of the IL-6 and IL-8 genes, and
under certain conditions each factor has been shown to activate both
proinflammatory genes (17, 18, 19, 20, 21, 22, 23, 24, 25). Furthermore, each of these
transcription factors has been implicated in IL-6 and IL-8 expression
in RA synovial tissue (26, 27, 28, 29). However, prior studies
have not directly examined, employing specific inhibitors, the
contribution of each of the transcription factors to the constitutive
and IL-1ß-stimulated expression of these genes by isolated
FLS.
In the present study, we employed adenoviral vectors expressing
dominant-negative (DN) versions of C/EBPß or c-Jun
(30, 31, 32, 33), and a nondegradable IB (34),
to determine the contribution of each transcription factor to the
constitutive and IL-1ß-stimulated expression of IL-6 and IL-8 by RA
FLS. More IL-6 and IL-8 was produced by RA FLS, both constitutively and
following IL-1ß stimulation, compared with control normal human
dermal fibroblasts (HDF). The IB-expressing adenovirus
significantly reduced the spontaneous and IL-1ß-stimulated expression
of IL-6 and IL-8 by the RA FLS, and of IL-1ß-stimulated HDF. The DN
C/EBPß resulted in modest reduction of the spontaneous and
IL-1ß-stimulated IL-6 secretion by RA FLS. In contrast, the DN
C/EBPß did not suppress the IL-1ß-stimulated IL-6 secretion by
HDF, nor did it affect IL-8 secretion by either cell type.
Inhibition of c-Jun had no effect in either IL-6 or IL-8, by RA FLS or
HDF, despite the fact that AP-1/c-Jun was strongly activated. The
inhibition of IL-1ß-stimulated IL-6 and IL-8 by the IB was due
to inhibition of NF-B activation, as determined by EMSA, and to
suppression of transcriptional activation. Supporting the critical role
of NF-B, mouse embryonic fibroblasts with both NF-B p50 and p65
genes deleted failed to express the IL-6 gene in response to IL-1.
These observations demonstrate that NF-B activation is the major
contributor to both IL-6 and IL-8 secretion by RA FLS, both
constitutively and following IL-1ß stimulation. These observations
support the role of inhibition of NF-B as a novel therapeutic target
in RA.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Normal HDF (CRL 1475) were purchased from American Type Culture Collection (ATCC, Manassas, VA). RA FLS were isolated from synovial tissue of patients undergoing scheduled total joint arthroplasty who met the American College of Rheumatology (former American Rheumatism Association) classification criteria for RA (35). Isolated synovial tissues were digested with collagenase, dispase, and DNAase I, and single-cell suspensions were obtained as previously described (36, 37). The cells were cultured at 37°C with 5% CO2 in DMEM supplemented with 10% FBS, 100 U/ml penicillin, 100 U/ml streptomycin, and 2 mM L-glutamine (all from Life Technologies, Gaithersburg, MD). The RA FLS were used between passages 4 and 10 and were free of contaminating cells (36, 37).
Viruses
Replication-defective adenovirus (Ad) vectors were propagated in
the 293 embryonic kidney cell line (ATCC) and purified by
ultracentrifugation through cesium chloride gradients. Plaque assay was
used for the determination of the titers of viral stocks. The
adenoviral vector AdIB (kindly provided by Dr. Jobin, University
of North Carolina) expresses a nondegradable form of human IB,
with hemagglutinin peptide added, in which serines 32 and 36 were
replaced by alanine residues (S32A/S36A), thereby blocking its
inducible phosphorylation and degradation (34, 38).
Adenoviral vectors expressing DN versions of C/EBPß (AdDNC/EBPß)
and c-Jun (AdDNc-Jun) were also employed. Each of these DNs lacked the
transactivation domain, but retained the ability to dimerize and bind
DNA, and inhibited the function of the respective wild-type
transcription factor (30, 31, 33, 39). Infection of
macrophages with either the AdDNC/EBPß or the AdDNc-Jun significantly
suppressed PMA-induced TNF- secretion by macrophages
(40).
Infections and collection of samples
A total of 1 x 104 cells/well were plated
in 24-well plates (Costar, Cambridge, MA) in DMEM 10%FBS, and allowed
to attach for 1–2 days. Cells were infected with AdIB,
AdDNC/EBPß, AdDNc-Jun, or the control vector expressing
ß-galactosidase (Adßgal) at a multiplicity of infection (moi) of
100 or 200 and incubated overnight. After washing twice with PBS, fresh
medium without or with 1 ng/ml of recombinant human IL-1ß (R&D
Systems, Minneapolis, MN) was added. After 48 h the supernatants
were collected for quantification of IL-6 and IL-8. All the experiments
were performed in triplicate.
EMSA
RA FLS (1.5 x 105 cells/100 mm
plate) were infected, and nuclear extracts were prepared 48 h
later, as previously described (31, 33), either following
IL-1ß (1 ng/ml) stimulation for 30 min or without IL-1ß treatment.
32P-labeled oligonucleotides containing the IL-6
B binding sequence
(5'-TCGACATGTGGGATTTTCCCATGAC-3'), the IL-8 B
binding sequence (5'-TCGAGCGTGGAATTTCCTCTGG-3'), the IL-6
C/EBPß binding sequence (5'-TCGAGACATTGCACAATCTG-3')
(41), or an AP-1 binding sequence from the collagenase
promoter (32), were used as probes for EMSA.
DNA-binding reactions were performed by incubation for 20 min at room
temperature in a final volume of 20 µl. The reaction mixture
contained 100 mmol/L NaCl, 20 mmol/L HEPES, 1 mmol/L EDTA, 4%
glycerol, 5% (w/v) Ficoll, 0.25 µg BSA, 1 µg poly(dI-dC), 1 ng
32P-labeled oligonucleotide, and 5–10 µg of
the nuclear extract. Protein:DNA complexes were separated from free
probe by electrophoresis on 5% polyacrylamide gels in 0.5x TBE at 160
V for 2–3 h. Gels were dried onto Whatman 3 M paper (Whatman,
Maidstone, U.K.) and exposed to Kodak XAR film (Eastman Kodak,
Rochester, NY). For supershift assays 1–2 µl of mouse mAb against
NF-B p65 (Transduction Laboratories, Lexington, KY), or rabbit
polyclonal Abs against NF-B p50 (NLS, sc114 X; Santa Cruz
Biotechnology, Santa Cruz, CA), NF-B p52 (K-27, sc-298 X; Santa Cruz
Biotechnology), c-Rel (N, sc70 X, Santa Cruz), C/EBPß (C-19, sc-150
X; Santa Cruz Biotechnology), or a goat polyclonal Ab against
c-Jun/AP-1 (N, sc-45 X; Santa Cruz Biotechnology), were incubated with
the nuclear extract on ice for 30 min before the addition of the
labeled oligonucleotide to the binding reaction.
Western blots
Cells were harvested, washed with PBS, to which lysis buffer
(150 mM NaCl, 0.5% Nonidet P-40, 50 mM Tris, 2 mM EDTA (pH 8.0))
containing protease inhibitors (2 ng/ml leupeptin, 0.5 mM PMSF, 15
ng/ml aprotinin) were added, to prepare cell extracts as previously
described (15, 42). Twenty micrograms of protein from each
sample was boiled and analyzed on 12.5% polyacrylamide gels for 3
h, transferred to polyvinylidene fluoride membranes (Immobilon P;
Millipore, Bedford, MA), and blocked with PBS containing 0.2% Tween
20/5% nonfat milk for 1 h. The membranes were incubated overnight
at 4°C with rabbit monospecific Abs to IB (C-21, sc 371; Santa
Cruz Biotechnology), C/EBPß (C-19, sc 150; Santa Cruz Biotechnology),
or c-Jun (PC06L; Oncogene Research Products, Cambridge, MA). Also, a
mouse anti-tubulin mAb (Calbiochem, La Jolla, CA) was used for the
examination of equal loading. The filters were washed three times with
PBS containing 0.2% Tween 20 and 5% nonfat milk and incubated for
2 h with donkey anti-rabbit or anti-mouse secondary Abs
(1:2000 dilution) conjugated to HRP (Amersham, Piscataway, NJ). After
washing, the blots were developed using enhanced ECL (Amersham).
RT-PCR
RNA was isolated by the RNAzol B method (Tel-Test, Friendwood, TX) as described by the manufacturer. One microgram of total RNA was incubated in reaction buffer containing oligo(dT) primer, avian myeloblastosis virus reverse transcriptase and RNase inhibitor (recombinant RNasin RNase inhibitor), and dNTP mixture for 1 h at 42°C. The reaction was stopped by incubation at 94°C for 5 min. PCR was performed using primers for IL-6 (forward 5'-ATGAACTCCTTCTCCACAAGCGC-3', reverse 5'-GAAGAGCCCTCAGGCTGGACTG-3') and for IL-8 (forward 5'-CCAAGGAAAACTGGGTGCAGAG-3', reverse 5'-GGCACAGGGGAACAAGGACTTG-3'), and ß-actin (Clontech, Palo Alto, CA). Cycling conditions included: one initial denaturation cycle for 5 min at 94°C, 25 cycles of amplification for 2 min at 72°C, 1 min at 94°C, 1 min at 60°C, and a final extension phase consisting of 1 cycle of 10 min at 72°C. Ten microliters of the PCR product and 2 µl of loading buffer were run on a 1% agarose gel stained with ethidium bromide.
RNase protection assay
Untreated and IL-1-stimulated wild-type and p65/p50-/- mouse embryonic fibroblasts (43) were harvested for RNA preparation employing Tri-Reagent (Molecular Research Center, Cincinnati, OH). RNase protection assay was performed according to the manufacturer’s specifications (PharMingen, San Diego, CA).
ELISA
For IL-6 and IL-8, sandwich ELISAs were performed, according to the manufacturer’s instructions, employing commercially available kits (DY206 and DY208 DuoSet kits; R&D Systems). The OD were read by a Microplate Autoreader (Bio-Tek, Burlington, VT).
Statistical analysis
To estimate the effect of IL-1ß stimulation or viral infections on the production of cytokines, the mean values of the experiments done with the cells of individual patients or HDF were analyzed using a two-tailed paired Student’s t test. To compare the levels of cytokines produced by HDF and RA FLS, an unpaired Student’s t test was used.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Normal HDF produced very low levels of IL-6 and IL-8
spontaneously, but the production increased significantly following
IL-1ß stimulation (IL-6 from 0.02 ± 0.02 ng/ml to 1.07 ±
0.41 ng/ml, p < 0.04; and IL-8 from 0.09 ± 0.05
ng/ml to 5.33 ± 0.85 ng/ml, p < 0.002). The
concentrations of the constitutively secreted IL-6 and IL-8 were
significantly (p < 0.005 and p
< 0.03, respectively) greater in the supernatants of the RA FLS
compared with the HDF (Fig. 1
). The
production of both cytokines by RA FLS increased significantly
following IL-1ß stimulation (IL-6 from 0.67 ± 0.19 to
11.58 ± 3.08 ng/ml, p < 0.03; and IL-8 from
1.44 ± 0.56 to 33.27 ± 7.50 ng/ml, p <
0.02). Following IL-1ß treatment, the concentrations of both
cytokines were greater in the supernatants of the RA FLS compared with
the HDF (p < 0.005). Thus, although the RA FLS
spontaneously secreted significantly more IL-6 and IL-8 than the HDF,
they remained highly responsive to IL-1ß.
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B and DNs of C/EBPß and
AP-1/c-Jun
Expression of the AdßGal was examined by x-Gal staining 48
h after infection. Although only about 50% of cells were positive at
100 moi, at 200 moi of AdßGal >95% of the HDF and RA FLS expressed
ß-galactosidase (data not shown). Because cell death was not observed
with any of the viruses at 200 moi, this concentration was used for
subsequent experiments. Western blot analyses using whole-cell extracts
of RA FLS and HDF demonstrated that DN proteins encoded by the viruses
(AdIB, AdDNC/EBPß, and AdDNc-Jun) were highly expressed (Fig. 2). EMSAs, using nuclear extracts of RA
FLS, were employed to document the function of each ectopically
expressed molecule (discussed below, see Figs. 6 and 7). The AdIB
inhibited the binding of NF-B to the IL-6 B (Fig. 6A)
and IL-8 B (data not shown) oligonucleotides, and the DN C/EBPß
and DN c-Jun bound to their respective oligonucleotides (see Fig. 7).
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The concentrations of IL-6 and IL-8 constitutively secreted by the
normal HDF were too low to characterize the effects of the
DN-expressing adenoviruses. Therefore, we examined the effect of these
vectors on the IL-1ß-stimulated secretion of IL-6 and IL-8 by HDF.
Compared with the control virus (Adßgal), infection of normal HDF by
AdIB resulted in >95% suppression of IL-1ß-stimulated IL-6
(p < 0.005) and IL-8
(p < 0.004) (Fig. 3). In contrast, infection of HDF with
AdDNC/EBPß or AdDNc-Jun had no effect on the production of either
IL-6 or IL-8 following IL-1ß stimulation. These findings suggest that
NF-B is essential for the IL-1ß-stimulated expression of both
cytokines by normal HDF.
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The effect of the DN-expressing adenoviral vectors on the IL-6 and
IL-8 constitutively secreted by RA FLS was examined. AdIB
infection resulted in significant suppression of the spontaneous
secretion of both IL-6 (51%, p < 0.001) and IL-8
(82%, p < 0.002) by RA FLS, compared with Adßgal
infection (Fig. 4). Expression of the DN
C/EBPß resulted in a modest but statistically significant reduction
of the spontaneous secretion of IL-6 (19%, p < 0.02),
compared with the control ß-galactosidase (Fig. 4A).
However, there was no difference in the concentration of IL-6 between
the AdDNC/EBPß-infected cells and those that were uninfected (Fig. 4A). In contrast, infection with the AdDNC/EBPß had no
effect on the spontaneous secretion of IL-8 by the RA FLS, compared
with Adßgal-infected or uninfected cells (Fig. 4B).
Additionally, expression of the DN c-Jun had no effect on constitutive
expression of either IL-6 or IL-8 by RA FLS. These observations
document that activation of NF-B is critical for the constitutive
expression of IL-6 and IL-8 by RA FLS. C/EBPß contributed to the
secretion of IL-6 but not IL-8, while AP-1/c-Jun did not play a
role.
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B, C/EBPß, and c-Jun on the
IL-1ß-stimulated secretion of IL-6 and IL-8 by RA FLS was examined.
Similar to the results observed with the HDF, the IL-1ß-stimulated
production of IL-6 by RA FLS was significantly inhibited by AdIB
(by 86%, p < 0.001) (Fig. 5A). In contrast to the
results obtained with the HDF, the DN C/EBPß resulted in a modest
reduction of IL-1ß-stimulated IL-6 (23%, p <
0.005), compared with the control ß-galactosidase (Fig. 5A). Similar to the results of the unstimulated cultures, no
difference was observed between the AdDNC/EBPß infected and the
uninfected RA FLS. Additionally, the ectopic expression of the DN c-Jun
had no effect on IL-1ß-stimulated IL-6 secretion.
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B infection, which
resulted in 90% (p < 0.001) reduction (Fig. 5
B). Expression of neither the DN C/EBPß nor the DN c-Jun
had any effect on IL-1ß-stimulated IL-8 secretion by RA FLS (Fig. 5B). These observations suggest that NF-B is the critical
transcription factor responsible for both the spontaneous and the
IL-1ß-stimulated expression of the IL-8 gene by RA FLS. Activation of wild-type transcription factors and the effect of DNs in RA FLS
To further characterize the mechanism of suppression observed in
this study, EMSAs were employed, using radiolabeled oligonucleotides
capable of binding NF-B, C/EBPß, and AP-1. NF-B, C/EBPß, and
c-Jun were each constitutively activated and bound to their respective
oligonucleotides, employing nuclear extracts from unstimulated RA FLS
(Figs. 6 and 7). Following stimulation with IL-1ß,
NF-B and c-Jun binding were each enhanced (Figs. 6 and 7). In
contrast, binding by C/EBPß was somewhat diminished following
stimulation with IL-1ß (Fig. 7A). In each instance,
specificity was demonstrated by inhibition with excess unlabeled
oligonucleotide and with monospecific Abs (Figs. 6 and 7). Monospecific
Ab to C/EBPß supershifted its respective complex (Fig. 7A). The AP-1 oligonucleotide-bound complex was partially
supershifted by monospecific anti-c-Jun, indicating that additional
factors may also be bound (Fig. 7B).
The complex binding to the IL-6 promoter NF-B binding site,
following treatment with IL-1ß, contained principally NF-B p65
because monospecific Abs to NF-B p65 resulted in a supershift of the
majority of the complex (Fig. 6A). No effect was observed
with anti-NF-B p52, or with c-Rel or the irrelevant control
(anti-c-Jun) Ab. Employing the monospecific anti-NF-B p50
Ab, supershift of a portion of the complex was observed (Fig. 6A). This same Ab caused a dramatic supershift of NF-B
p50 homodimers employing macrophage nuclear extracts (data not shown,
and Ref. 33). Combining Abs to p65 and p50 failed to
supershift the entire complex, suggesting the presence of an
unidentified component. These findings indicate that the IL-6 B site
bound mainly NF-B p65 and, to a lesser extent, NF-B p50.
Because the sequence of the IL-8 NF-B binding site was slightly
different from the IL-6 B site (see Materials and
Methods), we examined binding to the IL-8 B site. NF-B in
the RA FLS nuclear extracts bound constitutively, and the binding
increased following IL-1ß stimulation (Fig. 6B). Only the
Ab recognizing NF-B p65 inhibited the binding of the complex (Fig. 6B). Abs recognizing NF-B p50, c-Rel (Fig. 6B), and NF-B p52 (not shown) did not decrease binding of
the complex to the B IL-8 oligonucleotide. These observations
suggest that the NF-B p65 homodimers were the principal complex
contributing to the expression of the IL-8 promoter in RA FLS.
The effect of each of the inhibitory molecules was examined by EMSA,
employing nuclear extracts from RA FLS. Infection with the AdIB
inhibited spontaneous (data not shown) and IL-1ß-induced binding to
the IL-6 B (Fig. 6A) and IL-8 B (data not shown)
promoters. The expression of the DN C/EBPß (Fig. 6A) or
the DN c-Jun (data not shown) did not affect NF-B activation, as
determined by EMSA. Expression of the DN C/EBPß resulted in a
dramatic increase of the transcriptionally inactive version of C/EBPß
(Fig. 7A). This complex was also supershifted by the
monospecific Ab (data not shown). The bound DN C/EBPß overlapped with
the wild-type C/EBPß, precluding evaluation of the effect of the DN
on the wild type in this experiment. Previous studies have demonstrated
that this DN may inactivate the wild-type C/EBPß by heterodimerizing
with and inactivating wild-type C/EBPß or by binding as a homodimer,
displacing the wild type from its DNA binding site (31).
Expression of the DN c-Jun resulted in inhibition of binding by the
wild-type complex, with binding of the transcriptionally inactive
version of c-Jun, which migrated more rapidly in the gel (Fig. 7B). In contrast, the ectopic expression of the IB did
not effect the migration of the AP-1 binding complex (Fig. 7B). These data document the functional effects of IB,
DN C/EBPß, and c-Jun by EMSA.
IB suppresses the transcriptional activation of IL-6 and IL-8
in RA FLS
To further characterize the mechanism by which IB functions
in RA FLS, RT-PCR was performed (Fig. 8A). As expected, following
stimulation with IL-1ß, increased mRNA for both IL-6 and IL-8 was
observed. AdIB infection resulted in reduced IL-6 and IL-8 m-RNA
compared with infection with Adßgal, constitutively and following
treatment with IL-1ß (Fig. 8A). The control ß-actin was
expressed comparably following infection with either virus. These
observations indicate that infection with the AdIB results in
inhibition of NF-B activation and subsequent suppression of the
transcriptional activation of the IL-6 and IL-8 genes in RA
FLS.
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B is essential for IL-6 expression
Our observations (Figs. 4 and 5) and those of others (26, 44) suggest that factors other than NF-B may be necessary for
the expression of IL-6, at least in RA FLS. To determine whether
NF-B was necessary for IL-6 expression, we employed mouse embryonic
fibroblasts in which both NF-B p50 and p65 genes had been deleted.
Following stimulation with IL-1, the IL-6 gene was expressed in
wild-type mouse embryonic fibroblasts, as determined by an RNase
protection assay (Fig. 8B). In contrast, no expression of
the IL-6 gene was detected following IL-1 stimulation of cells lacking
the NF-B p50 and p65 genes. These observations indicate that NF-B
activation is essential for the expression of the IL-6 gene mouse
embryonic fibroblasts, supporting the observations obtained with the
normal HDF (Fig. 3).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B complexes binding the IL-6 and IL-8 promoters was somewhat
different, expression of IB inhibited the nuclear localization
and activation of all IL-6 and IL-8 B binding complexes.
Additionally, expression of the IB resulted in suppression of the
transcription of both the IL-6 and IL-8 genes and in the marked
reduction of the constitutive and IL-1ß-stimulated secretion of IL-6
and IL-8 by RA FLS. The observed results were not due to decreased cell
numbers or apoptosis (data not shown). These observations document the
critical role of NF-B in the expression of IL-6 and IL-8 by RA
FLS.
Other studies have examined the potential contribution of NF-B to
the expression of IL-6 and IL-8 by RA FLS. NF-B activation was
temporally associated with IL-6 and IL-8 expression in RA FLS (4, 26, 29, 45, 46). Inhibition of NF-B by
N-acetyl-L-cysteine or aurothioglucose
was associated with inhibition of IL-6 and IL-8 secretion by RA FLS
(45, 46). Although these observations suggest that NF-B
activation may be related to the expression of IL-6 and IL-8 by RA FLS,
none has specifically defined the role of NF-B in isolated RA FLS.
Our study is novel because it has directly documented the contribution
of NF-B to the constitutive and the IL-1ß-stimulated secretion of
IL-6 and IL-8, employing isolated RA FLS. Employing whole RA synovial
tissue digests possessing fibroblasts, macrophages, and T cells, the
constitutive secretion of IL-6 and IL-8 was inhibited by 85% and 40%,
respectively, by a porcine IB-expressing adenovirus
(47). However, these results are difficult to compare with
ours because the porcine IB employed was not modified to prevent
degradation following activation, and cytokines, including TNF- and
IL-1, were present in the culture supernatants. Additionally, both
macrophages and FLS from RA synovial tissue produce IL-6 and IL-8, and
this study did not document the cell type(s) responsible, nor did it
address the potential contribution of other transcription factors
(47). Nonetheless, both observations, using different
experimental conditions, have documented the important role of NF-B
in the expression of IL-6 and IL-8 in RA synovial tissue.
The role of C/EBPß and AP-1/c-Jun in the expression of IL-6 and IL-8
in RA FLS was also examined. Earlier studies have documented that
C/EBPß and AP-1 may contribute to the activation of the IL-6 and IL-8
genes (19, 20, 21, 28). Our study employed a novel approach to
specifically inhibit the function of each potentially relevant
transcription factor. Neither C/EBPß nor AP-1/c-Jun contributed to
the expression of the IL-8 gene, in either RA FLS or HDF, despite the
activation of both C/EBPß and AP-1 in RA FLS. It is possible that
C/EBPß or c-Jun may contribute to the expression of IL-8 in other
cell types or when the cells are stimulated differently (24, 48). Nonetheless, our observations do not exclude a potential
interaction of NF-B with AP-1 or C/EBPß (19, 20, 21, 24, 28, 48). Because we did not employ AdDNC/EBPß and AdDNc-Jun
simultaneously, it is possible that the expression of the IL-8 gene may
require NF-B plus either C/EBPß or AP-1 (20, 24, 25).
Although the expression of IL-6 in RA FLS was regulated primarily by
NF-B, the DN C/EBPß resulted in a 20% reduction, both
spontaneously and following IL-1ß stimulation, suggesting that
C/EBPß contributes to the activation of the IL-6 gene in this cell
type. However, an alternate interpretation, which cannot be excluded
from our data, is that adenovirus infection resulted in increased IL-6,
but not IL-8, and that this increase was due to C/EBPß. Arguing
against this possibility, adenoviral infection had no effect on the
IL-6 secreted by the HDF, and no activation of C/EBPß was observed
following adenoviral infection of RA FLS (Fig. 7A). These
observations are consistent with previous studies showing that C/EBPß
was not necessary for the expression of the IL-6 gene, because
C/EBPß-/- knockout mice produced essentially
normal IL-6 (49, 50). Following stimulation with IL-1ß,
inhibition of NF-B and C/EBPß accounted for essentially all of the
IL-6 secreted. Although NF-B and C/EBPß have been shown by
transient transfection to synergistically activate the transcription of
the IL-6 gene (22), the approach employed in this study
does not allow us to determine whether or not these two factors were
interacting synergistically in RA FLS. In contrast to the results
observed with the RA FLS, employing normal fibroblasts, only NF-B
contributed to IL-1-stimulated IL-6 activation, because IB in the
HDF and deletion of NF-B p50 and p65 in mouse embryonic fibroblasts
essentially abolished IL-1-stimulated IL-6 gene expression.
An earlier study has suggested that AP-1 may contribute to IL-6 and
IL-8 secretion by RA FLS, because jun D down-modulated AP-1
and suppressed TNF--induced secretion of these cytokines
(28). In contrast, our study clearly demonstrated that
even though AP-1 activation was suppressed by EMSA following expression
of the DN c-Jun, no suppression of IL-6 or IL-8 secretion was observed.
Activation of the mitogen-activated protein (MAP) kinase pathway has
been shown to contribute to both IL-6 and IL-8 expression (44, 51, 52). Inhibition of the p38 MAP kinase pathway in RA FLS, or
the extracellular signal-related kinase-1/2 pathway in monocytes,
suppressed IL-6 secretion without affecting NF-B activation
(44, 51). In RA FLS p38, MAP kinase activation by IL-1ß
contributed to IL-6 expression by stabilizing the IL-6 mRNA
(44). Similarly, MKK6, a p38 MAP kinase-specific
activator, stabilized IL-8 mRNA, while activation of the c-Jun
N-terminal kinase pathway by MKK7 enhanced IL-8 synthesis and IL-8
promoter activity (52). Despite the effects of MKK6 and 7
on the regulation of the IL-8 gene, basal NF-B activation was always
present, suggesting that activation of the MAP kinase pathway may
enhance NF-B-induced IL-8 expression. Although these observations
suggest that activation of the p38 and extracellular signal-related
kinase-1/2 MAP kinase pathways may modulate IL-6 and IL-8 expression,
our observations demonstrate that activated AP-1/c-Jun did not
participate in the spontaneous or IL-1ß-stimulated activation of the
IL-6 or IL-8 genes in either RA FLS or HDF. However, because the
activation of NF-B observed by EMSA was comparable for both cell
types (data not shown), it is possible that modulation of gene
expression by MAP kinase pathway activation may have contributed to the
differences observed between the RA FLS and the HDF, by mechanisms that
do not involve the AP-1 complex.
The composition of the B complexes responsible for the
IL-1ß-stimulated activation of the IL-6 and IL-8 promoters in RA FLS
were somewhat different. NF-B p65 was the most abundant species
binding to the IL-6 B site, while Abs to NF-B p50 partially
supershifted the IL-6 B binding complex. These observations suggest
the presence of p65 homodimers and p65/p50 heterodimers following
IL-1ß-stimulation, consistent with earlier observations
(26). Although controversy exists in the literature
(21, 23), employing RA FLS, only NF-B p65 bound to the
IL-8 B oligonucleotide, and no NF-B p50 containing heterodimers
or p50 homodimers were observed. In a previous study, using RA FLS,
antisense oligonucleotides to NF-B p65, and c-Rel, but not NF-B
p50, partially inhibited the IL-1ß-stimulated IL-8 secretion by RA
FLS, suggesting a potential role for c-Rel (53). However,
similar to our observations, no c-Rel-containing complexes were
identified (53). Additionally, the effectiveness and
specificity of the c-Rel antisense oligonucleotides employed in this
study were not documented, making the interpretation that c-Rel
contributed to the expression of the IL-8 gene in RA FLS inconclusive
(53). A recent study employing rabbit FLS identified a
major contribution of NF-B p50 to the regulation of
IL-1ß-stimulated matrix metalloproteinase 1 (54). In
contrast, no NF-B p50 homodimers were detected with RA FLS nuclear
extracts employing the IL-6 B oligonucleotide, or an HIV/Ig NF-B
binding oligonucleotide, which avidly bound p50 homodimers in
macrophages (Ref. 33 , and data not shown). These
observations document that NF-B p65 was the dominant species binding
to both IL-6 and IL-8 B binding sites, although NF-B p65/p50
heterodimers also bound to the IL-6, but not the IL-8, B site, in
IL-1ß-treated RA FLS nuclear extracts. Together, these observations
identify the central role of NF-B in the regulation of the IL-6 and
IL-8 genes by RA FLS, and they suggest that inhibition of NF-B may
be an effective target in the treatment of RA.
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Acknowledgments |
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B. |
Footnotes |
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2 Current address: Department of Rheumatology, 251 Hellenic Air Force and Veterans Administration General Hospital, P. Kanellopoulou 3 Street, Athens, Greece.
3 Address correspondence and reprint requests to Dr. Richard M. Pope, Division of Rheumatology, Department of Medicine, Northwestern University Medical School, 303 East Chicago Avenue, Ward 3-315, Chicago, IL 60611.
4 Abbreviations used in this paper: RA, rheumatoid arthritis; FLS, fibroblast-like synoviocytes; HDF, human dermal fibroblasts; DN, dominant-negative; moi, multiplicity of infection; Ad, adenovirus; MAP, mitogen-activated protein.
Received for publication April 5, 2000. Accepted for publication September 13, 2000.
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