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-Induced Production of IL-6 and IL-8 from Cultured Synovial Fibroblasts1
Departments of
*
Molecular Genetics and
Orthopedics, Nagoya City University Medical School, Nagoya, Japan; and
Louis Pasteur Center for Medical Research, Kyoto, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B. We found that the serum TRX
concentration was elevated in patients with rheumatoid arthritis (RA)
as compared with values from healthy individuals and patients with
osteoarthritis (33.6 ± 35.1 vs 11.8 ± 6.6 ng/ml,
p < 0.01). Moreover, the TRX concentration in the
synovial fluid (SF) was much more elevated in RA patients than in
osteoarthritis patients (103.4 ± 53.3 vs 24.6 ± 17.4 ng/ml,
p < 0.001). Multiple regression analysis revealed
that the serum C-reactive protein value was better correlated with the
linear combination of SF TNF-
and SF TRX values than with SF TNF-
alone, suggesting that TRX might play a subsidiary role in the
rheumatoid inflammation. We thus examined the effect of TRX on the
TNF--induced IL-6 and IL-8 production using rheumatoid synovial
fibroblast cultures. The extents of IL-6 and IL-8 production in
response to TNF- were greatly augmented by TRX as compared with
TNF- alone. TRX alone did not have such effects. We also found that
TRX appeared to accelerate the nuclear translocation of NF-B, a
major transcriptional regulator for production of IL-6 and IL-8 on
stimulation with TNF-. Consistent with these findings, the IB
phosphorylation at Ser32 and its subsequent degradation in
response to TNF- was facilitated by TRX. These findings indicate
that the elevated TRX concentration in SF of RA patients might be
involved in the aggravation of rheumatoid inflammation by augmenting
the NF-B activation pathway. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cytokines that are abundantly produced in the inflamed rheumatoid
synovial fluid include TNF-, IL-1, IL-6, IL-8, and GM-CSF (1, 4, 6, 7, 8). Among these cytokines, TNF- and IL-1 are considered to play
crucial roles because they are known to induce IL-6, IL-8, GM-CSF, and
themselves (5, 6, 9, 10). Production of CRP in the liver is stimulated
by IL-6 (11, 12, 13), and the serum concentrations of IL-6 and CRP were
well correlated (4, 14). Additionally, expression of some cellular
adhesion molecules, including ICAM-1, VCAM-1, and E-selectin, that are
responsible for accumulation of the inflammatory cells in the inflamed
tissue, can be induced by TNF- and IL-1 (9, 15, 16, 17). TNF- and
IL-1 stimulate inducible expression of these genes through a signal
transduction pathway leading to activation of cellular transcription
factors such as NF-B (18, 19, 21). In concordance with these
observations, Handel et al. (22) and Marok et al. (23) have
demonstrated the nuclear localization of NF-B in the synovial lining
cells of freshly isolated tissues from RA patients, indicating the
activation of NF-B in situ. It was also noted that some of the drugs
effective in the treatment of RA have recently been demonstrated to
have inhibitory actions on the NF-B activity or its activation
cascade (10, 24, 25, 26, 27, 28).
NF-B is an inducible transcription factor present in primordial
mesenchymal cell lineage including lymphocytes, macrophages, and
fibroblasts (19, 22, 23). NF-B is composed of two subunits, p50 and
p65, and exists as an inactive form in the cytoplasm associated with
the inhibitory molecule IB. On stimulation by proinflammatory
cytokines such as TNF- and IL-1, NF-B is dissociated from IB
in association with its proteolytic degradation and then moves into the
nucleus (29), where it activates target genes including IL-6, IL-8,
ICAM-1, VCAM-1, and E-selectin (18, 19, 21). Although NF-B is by no
means the sole determinant for inducible expression of these genes,
molecular genetic studies, such as elucidations of the responsible
cis-regulatory elements in promoter regions of inducible
expression of these genes, have indicated that NF-B plays a
indispensable role (18, 19, 20, 21).
It has been assumed for years that oxidative stress is involved in the pathophysiology of RA. Activated macrophages in the inflamed RA joint were considered to be responsible for the production of radical oxygen intermediates (ROI) (30). The increase in intraarticular pressure (31, 32, 33) and the subsequent hypoxic-reperfusion injury in synovial tissue was considered to generate ROI owing to the uncoupling of intracellular redox systems (32). These were evidenced by demonstration of the increase in lipid peroxidation products (32), the presence of ROI-mediated degradation of hyaluronic acid (33), and depletion of ascorbate (34) in synovial fluids of patients with RA.
NF-B is known to be regulated by the redox mechanism (9, 35, 36, 37, 38). We
and others have demonstrated that the DNA-binding activity of NF-B
is regulated by an oxidoreductive mechanism ("redox regulation")
where ROI and the cellular reducing catalyst thioredoxin (TRX) play
major roles (35, 36, 37, 38). Qin et al. (37) revealed the formation of a
molecular complex between TRX and the DNA-binding loop of NF-B p50
subunit by NMR. Accumulating evidence indicates that ROI are involved
in the initial stage of the NF-B activation cascade (9, 38, 46, 54).
Interestingly, TRX production is known to be induced by ROI (39, 49),
although the precise actions of ROI and the molecular mechanism of ROI
production in response to TNF- or IL-1 are not clarified.
TRX is thus considered to be a signaling effector for the activation of
NF-B (35, 36, 38). It was noted in earlier studies that TRX
synergized the effects of IL-1 in promoting thymocyte growth (40) and
in inducing IL-2 receptor -chain expression (41). Moreover, Schenk
et al. (42) found that TRX could act as a costimulus in the inducible
expression of those cytokines that are under the control of NF-B.
These observations led us to investigate the role of TRX in the
pathogenesis of RA and to examine its effect on the production of those
cytokines responsible for rheumatoid inflammation such as IL-6 and IL-8
from the cultured synovial fibroblasts obtained from patients with RA.
We have also examined the relationships among the concentrations of TRX
and TNF- in the SF and the concentration of serum CRP as a surrogate
marker of rheumatoid inflammation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Seventy-one RA patients whose diagnosis were defined by the 1987 revised the American College of Rheumatology (formerly the American Rheumatism Association) criteria (43) were enrolled in this study. Patients (53 women and 18 men) ranged in age from 35 to 80 yr with a mean age of 61.7 ± 11.3 yr. The mean disease duration was 14.3 ± 11.3 yr with a range of 1–37 yr. Seventeen patients (12 women and 5 men) with osteoarthritis (OA) (mean age, 74.0 ± 7.6 yr; mean disease duration, 6.5 ± 5.8 yr) were also enrolled in this study as a control. The average age of 54 healthy controls (23 women and 31 men) were 47.6 ± 15.1 yr (range, 25–70 yr). Informed consent was obtained from each patient in conformity with requirements of the ethics committee at Nagoya City University Medical School.
Synovial fluid and serum
SF was aspirated from knee joints, collected into a sterile vials containing heparin, immediately centrifuged at 600 x g for 15 min to remove blood cells at 4°C, and stored at -80°C. Serum samples were obtained by venipuncture followed by centrifugation and stored at -80°C. To exclude the influence of cell lysis on TRX concentrations, a correction procedure was applied based on the hemoglobin (Hb) content in each sample according to the method of Nakamura et al. (44).
Cells
Rheumatoid synovial fibroblasts (RSFs) were isolated from the
fresh synovial tissue biopsy samples of three RA patients as previously
reported (9, 10, 45). Tissue samples were minced into small pieces and
treated with 1 mg/ml collagenase/dispase (Boehringer Mannheim,
Mannheim, Germany) for 10–20 min at 37°C. The cells obtained were
cultured in F-12 (Ham’s) (Life Technologies, Grand Island, NY)
supplemented with 10% FCS (Irvine Scientific, Santa Ana, CA), 100 U/ml
penicillin, 100 µg/ml streptomycin, and 0.5 mM mercaptoethanol. The
culture medium was changed every 3–5 days, and nonadherent lymphoid
cells were removed. Adherent cell subcultures were maintained in the
same medium and harvested by trypsinization every 7–10 days before
they reached cellular confluency. All the experiments described here
were conducted using the RSF during the 3rd-13th passage. Most of the
RSF cultures used were between the 5th and 7th passages. Only one RSF
culture of the 13th passage was used. As far as we tested for cytokine
induction and the sensitivity for NF-B nuclear translocation, no
significant difference was noticed among different RSF passages. To
characterize the phenotype of adherent cells, the cells were stained
with mouse mAbs against human HLA-DR, von Willebrand factor, desmin,
smooth muscle -actin, CD1, CD68, and 5B5. Only 5B5 was positive
for RSF, suggesting its fibroblast-like phenotype.
Reagents
Recombinant human TNF- was purchased from Boehringer
Mannheim. Purified recombinant human TRX is a generous gift from Dr A.
Mitsui (Institute of Basic Science, Ajinomoto, Kawasaki, Japan). Rabbit
polyclonal Ab to human NF-B subunits p65, human IB and its
phosphorylated form were purchased from Santa Cruz Biotechnology (Santa
Cruz, CA) and New England Biolabs (Beverly, MA), respectively. LPS in
the purified TRX preparation was measured with a commercial detection
kit (Endotoxin test-D, Seikagaku, Tokyo, Japan) and carefully removed
with polymyxin B (Affi-Prep Polymyxin Matrix, Bio-Rad, Richmond,
CA).
Cytokine assays
Amounts of cytokines produced in the cell culture supernatant
were measured by ELISA with serial dilutions from supernatants of cells
that were stimulated with or without various concentrations of TNF-
in the presence or absence of TRX. The cell culture supernatants were
harvested after stimulation with TNF- 1 h after treatment with
or without TRX and then stored at -80°C. The ELISA kits used were
the Biotrak human IL-6 and TNF- ELISA system (Amersham, Little
Chalfont, U.K.) and the Cytoscreen human IL-8 ELISA system (BioSource
International, Camarillo, CA), and measurements were performed
according to the manufacturers’ protocol.
TRX ELISA
Measurement of the TRX concentration was performed by sandwich ELISA as described by Nakamura et al. (44) with two murine mAbs to nonoverlapping epitopes of human TRX (ADF-11 and ADF-21) kindly provided by Fuji Rebio (Tokyo, Japan). Briefly, samples were added to the 96-microwell plates that were coated with ADF-21, incubated at room temperature for 2 h, and washed; HRP-labeled ADF-11 was added followed by incubation at room temperature for 2 h. After washing, a buffer containing substrate for HRP (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) and hydrogen peroxide was added and incubated at room temperature for 1 h. The absorption at 405 nm was measured on a ELISA reader (Vmax kinetic microplate reader, Molecular Devices, Menlo Park, CA). Recombinant human TRX was used as a standard. The Hb concentrations in serum and SF were measured with a commercial diagnostic kit according to the procedure described by the supplier (Sigma, St. Louis, MO). To correct the effect of hemolysis on TRX measurement, corrected TRX concentrations were calculated using the formula previously published (44): corrected TRX = TRX measured - 0.937 x Hb content.
Although some patient samples contained rheumatoid factor (RF), there was no apparent interference of RF in measuring TRX or any correlation between RF titers and TRX concentrations in these samples.
Immunofluorescence
Approximately 0.8 x 104 RFSs were
seeded and cultured in four-well LabTek chamber slides (Nunc,
Naperville, IL) and allowed to adhere for 72 h. The cells were
first incubated with or without 100 ng/ml TRX and then stimulated with
various concentrations (0, 10, 100, or 250 pg/ml) of TNF- for 30
min. Indirect immunofluorescence with the specific anti-p65 Ab was
performed as follows. The cells were fixed in PBS containing 4.5%
paraformaldehyde for 10 min at room temperature and permeabilized by
0.5% Triton X-100 in PBS for 20 min at room temperature. They were
incubated with rabbit polyclonal Ab against p65 subunit at 1:100
dilution for 45 min at 37°C. After a washing with PBS, the cells were
incubated with the second Ab, the FITC-conjugated goat anti-rabbit
(whole IgG) Ab (Cappel Organon Teknika, Durham, NC), for 20 min at
37°C (9, 10, 45). No staining was observed either when no primary Ab
was added or when normal rabbit serum instead of the primary Ab
was used.
Western blot analysis
RSF at 80% confluency growing in 60-mm culture dishes in F-12
containing 10% (v/v) FCS were stimulated with TNF- (500
pg/ml) and/or TRX (100 ng/ml). Treated cells were washed twice with
cold PBS and lysed in 0.2 ml ice-cold extraction buffer (20 mM
HEPES-KOH (pH 7.9), 350 mM NaCl, 20% glycerol, 1% Nonidet P-40, 1 mM
EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF, 20 mM ß-glycerol-P, 0.1 mM
Na3VO4, and 1 µg/ml each
of aprotinin, pepstatin, and leupeptin) on the rocker at 4°C for 30
min. Cells were scraped, the solubilized cell homogenates were
harvested and centrifuged at 10,000 x g for 30 min at
4°C, and the resultant supernatants were used as whole cell extracts.
Protein contents were determined with the DC Protein Assay kit
(Bio-Rad). Protein (14 µg of each sample) was loaded per lane on an
SDS-polyacrylamide gradient (5–20%) gel. Proteins were separated
electrophoretically and transferred to nitrocellulose membranes
(Hybond-C, Amersham, U.K.). For immunoblotting, membranes were blocked
with 5% nonfat dried milk in PBS containing 0.05% Tween 20 (TPBS) for
1 h at room temperature. The membranes were incubated with rabbit
polyclonal Ab against human IB (Biolabs) at 1:2000 dilution or
with rabbit polyclonal IgG Ab against phosphorylated IB
(Ser32) (Biolabs) at 1:1000 dilution
in TPBS containing 1% BSA for 1 h at room temperature. After
three washings in TPBS, membranes were further incubated for 1 h
at room temperature with peroxidase-conjugated goat anti-rabbit IgG
(Sigma) at 1: 2000 dilution in TPBS for 1 h and were washed in
TPBS three times before visualization of proteins. An enhanced
chemiluminescence ECL kit with Hyperfilm MP (Amersham) was used for
detection.
Statistical analysis
Statistical evaluation of differences in production of TRX,
IL-6, and IL-8 cytokine was performed by the unpaired Student’s
t test. We also applied Welch’s unpaired t test
to confirm the evaluations of the statistical difference of two groups
with different variations. In the situation where data distribution was
deviated from the normal distribution such as serum TRX concentrations,
Wilcoxon’s rank sum test was applied. Factors affecting serum CRP
values were investigated by multiple regression analysis using
Statistica for Windows Rel.5.1J (StatSoft, Tulsa, OK). The serum CRP
concentration was used as the objective variable, and the TNF- and
TRX concentrations in SF were used as explanatory variables. Student’s
t test was used to evaluate the partial regression
coefficient. Cluster analyses of serum CRP, SF TNF-, and TRX values
were performed by the K-means method. These multivariate analyses were
conducted with a computer model GXMT5200 (Dell, Round Rock,
TX).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The TRX concentrations in serum and SF were measured by ELISA. As
shown in Fig. 1
A, the serum
TRX concentration of 51 RA patients was significantly elevated as
compared from 64 healthy individuals (33.6 ± 35.1 vs 11.8 ±
6.6 ng/ml, p < 0.01 by Wilcoxon’s rank sum test). We
also measured TRX in the SF of 43 patients with RA and compared them
with those in SF of 17 patients with OA. As demonstrated in Fig. 1B, the SF TRX concentrations in RA patients were
significantly elevated from those of OA by a factor of 4 (127.7 ±
75.7 vs 22.5 ± 16.0 ng/ml, p < 0.001). In
addition, the TRX concentration appeared to be much greater in SF than
in the serum of RA patient. Because samples in Fig. 1, A and
B, were obtained at different occasions from these RA
patients, we analyzed TRX concentrations in the serum and SF that were
simultaneously obtained from RA patients. In Fig. 1C, TRX
concentrations in the paired serum and SF samples obtained from 20 RA
patients were compared. Those patients whose SF and serum samples were
not simultaneously available were omitted from this analysis. In all 20
RA cases, TRX concentrations in SF were greater than those in sera
(158.4 ± 74.4 vs 43.2 ± 44.5, p < 0.01),
which was consistent with the findings of Maurice et al. (48). However,
TRX concentrations in SF and those in sera were not significantly
correlated.
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, and
serum CRP in RA patients
We also measured the TNF- concentration in SF of 43 RA patients
and 17 OA patients. As shown in Fig. 2A, the SF TNF-
concentration was greatly elevated in RA as compared from OA
(p < 0.001). We then examined the
relationships of SF TNF- and TRX concentrations with the serum CRP
concentration, i.e., a widely used surrogate marker for systemic
inflammatory responses such as RA (Fig. 2, B and
C). Although there was a positive correlation between the SF
TNF- concentration and the serum CRP concentration in RA cases
(r = 0.421, p < 0.005) (Fig. 2B), no correlation was observed between the SF TRX and the
serum CRP (Fig. 2C) or between the serum TRX and the serum
CRP concentrations (data not shown). Additionally, there was no
correlation between the TRX and TNF- concentrations in SF of the RA
cases studied (Fig. 2D). Because of the presence of
interrelationships among these distinct parameters, as described in
Fig. 2, we performed multivariate analyses.
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The above observations based on simple statistics indicated that
there were cross-correlations among serum CRP, SF TRX, and SF TNF-
values. To delineate the genuine effects of SF TRX and SF TNF-
on the serum CRP concentration, we applied multiple
regression analysis using the observed values from 41 of 43 RA patients
whose SF and serum samples were obtained within 2-wk intervals. As
shown in Table I, serum CRP was
positively affected by TNF- and TRX with a multiple regression
coefficient (R) of 0.533 (p <
0.001). A partial regression coefficient for serum CRP concentration
was significant for SF TNF- (p < 0.001 by
Student’s t test) but not for SF TRX
(p > 0.1) as expected from the results in
Fig. 2
.
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illustrates a three-dimensional
scatter plot of the data from 41 RA patients, which indicated the
presence of two groups (Fig. 3). We then performed cluster analysis
using K-means method and classified the subjects into two distinct
groups that were characterized by lower (<40.1 pg/ml) (25 cases) and
higher (>40.1 pg/ml) SF TNF- (16 cases) concentrations (closed and
open circles for lower and higher SF TNF- groups, respectively, in
Fig. 3). When correlation between SF TRX and serum CRP was examined in
each group, there was a significant correlation between SF TRX and
serum CRP in those RA patients with lower SF TNF- (r
= 0.545, p < 0.01). However, no correlation was
observed in those RA patients with higher SF TNF-.
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These features of SF TRX indicated its role as a costimulatory
factor for the actions of TNF- in induction of inflammatory
responses. To determine how TRX is involved in the inflammatory
responses elicited by TNF-, we examined the effect of TRX on
cytokine production from the cultured RSFs according to methods
previously described (9, 10, 45). Because IL-6 is considered to be
involved in the production of CRP in the liver, we first examined
whether TRX could augment the IL-6 production from RSFs. As
demonstrated in Fig. 4A,
augmentation of IL-6 production by addition of TNF- was observed in
a dose-dependent manner for the amount of TNF-. The concentrations
of TNF- used in this experiment were based on the previous report
(9, 10) of TNF- concentrations in SF RA (50–250 pg/ml) and on our
measurement of the SF RA samples enrolled in this study (43.6 ±
27.9 pg/ml) (Fig. 2A). When increasing amounts of TRX was
added together with TNF-, further augmentation of IL-6
production was observed. For example, when 100 ng/ml TRX (corresponding
to the average TRX concentration in SF RA, Fig. 1B) were
added 1 h before addition of 100 pg/ml TNF-, the IL-6
production was greatly augmented as compared with that from the culture
without TRX with the same amount of TNF- (1.33 ± 0.257 vs
0.776 ± 0.078 ng/ml, p < 0.01). However, when
TRX was added without TNF-, no increase in IL-6 production was
observed. When TRX was added simultaneously or 1 h after the
addition of TNF-, no such effect on IL-6 production was
demonstrated, and the greatest effect of TRX was obtained when TRX was
added 1 h before addition of TNF- (data not shown). Similarly,
IL-8 production in response to TNF- was greatly augmented by the
addition of TRX 1 h before adding TNF- (Fig. 4B).
Again, no effect was observed when TRX was added alone or
simultaneously with TNF-.
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-induced nuclear translocation of
NF-B
Because IL-6 and 8 have NF-B-binding sites in their promoter
regions and are under the transcriptional control of NF-B (19, 47),
we examined whether TRX could facilitate the TNF--induced activation
of NF-B. Indirect immunostaining using Ab to the p65 NF-B subunit
was conducted with RSF cultures stimulated with various concentrations
of TNF-. Nuclear translocation of NF-B was demonstrated after 30
min of treatment with TNF- at concentrations >10 pg/ml. However,
when 100 ng/ml TRX (an average TRX concentration in SF RA) were added
1 h before TNF addition, the NF-B nuclear translocation was
significantly promoted (Fig. 5A). As shown in Fig. 5B, the percent nuclear translocation of NF-B was
augmented by addition of TRX in the presence of 10 pg/ml TNF-
(48.7 ± 7.9% vs 23.3 ± 6.3%, p
< 0.01). TRX alone, even at high concentrations (we examined up to 100
µg/ml), could not induce NF-B nuclear translocation at all.
Similar experiments were performed with the use of RSF cultures
obtained from other RA patients. Essentially, the similar results were
obtained (data not shown).
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B
Considering the roles of IB in NF-B activation pathway in
which it retains NF-B in the cytoplasm by masking the nuclear
localization sequence, we investigated the amount of IB as well
as its phosphorylation status at the regulatory amino acid residue
(Ser32) by Western blotting using specific Abs.
To explore the mechanism by which TRX increases the nuclear
translocation of NF-B (as shown in Fig. 5), we determined
whether TRX could accelerate the TNF--induced IB
phosphorylation and degradation. Whole cell extracts were obtained from
the cultured RSF after treatment with TNF- (after 0, 5, or 15 min)
with or without TRX pretreatment and were analyzed by Western blotting
using Abs against IB or its phosphorylated form at
Ser32. Fig. 6
demonstrates that in the RSF treated with TNF- alone, the IB
concentration was retained at 5 min and then decreased after 15 min of
the treatment. On the other hand, IB degradation was observed
more rapidly after 5 min of the treatment with TNF- together with
TRX (100 ng/ml), and most of the protein band corresponding to IB
disappeared after 15 min. Furthermore, as shown in the middle panel of
Fig. 6, the augmented phosphorylation of IB was detected after 5
min of the TNF- stimulation in the presence of TRX as compared from
the treatment with TNF- alone. The appearance of the phosphorylated
form of IB was detected even at 5 min after stimulation with
TNF- before the degradation of IB. These findings indicated
that TRX could augment the phosphorylation of IB and its
degradation that were elicited by TNF-.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in determining the serum CRP concentration.
These results indicated that TRX might serve as a cofactor for TNF-
in inflammatory responses. To demonstrate such actions of TRX, we
applied a cell culture model and examined the effect of TRX on the
production of inflammatory cytokines, IL-6 and IL-8, that were
stimulated by TNF-. Using cultured RSFs, we were able to demonstrate
that TRX could augment the TNF--induced up-regulation of IL-6 and
IL-8 production. We also demonstrated that this effect of TRX might be
through the upstream signal transduction pathway leading to the
phosphorylation of IB as well as its subsequent degradation and
thus promoting the nuclear translocation of NF-B, a major
determinant of the induced production of IL-6 and IL-8. Consistent with our observations, Maurice et al. (48) reported that TRX was increased in the SF of RA patients and claimed that this increase of TRX could be the result of an increase in oxidative stress in SF (31, 32, 33, 34). Although the range of SF TRX concentration reported by Maurice et al. (48) was higher (282 ± 42 ng/ml) than ours, this could be ascribed to the difference of the assay system used. Nakamura et al. (44) reported the increase of TRX in sera of HIV-infected patients. They observed a negative correlation between serum TRX and CD4 count, suggesting the presence of oxidative stress in the advanced HIV infection. Taken together, these reports indicated that TRX could serve as a marker for oxidative stress in vivo.
It was shown that TRX was produced in response to oxidative stress.
Sachi et al. (39) demonstrated the induced production of TRX in human
keratinocytes by UVB irradiation or treatment with hydrogen peroxide.
Moreover, Taniguchi et al. (49) identified a responsible
cis-regulatory element in response to oxidative stress
within a promoter region of human TRX gene. Thus, the increase of TRX
concentration in SF could be ascribed to the production of ROI by
activated macrophages and by hypoxic-reperfusion injuries in the
inflamed rheumatoid joints as discussed earlier (31, 32, 33, 34). The increase
in TRX concentration in sera of RA patients (Fig. 1), although to a
lesser extent, could be secondary to the increase of SF TRX.
Regarding the action of TRX, there are various reports supporting our
experimental findings of augmentation of the TNF--induced IL-6 and
IL-8 production (Fig. 4). For example, earlier studies on TRX such as
that of Wakasugi et al. (40) reported that TRX (3B6-IL-1) showed
IL-1-like activities. Similarly, Tagaya et al. (41) reported that TRX
could induce the IL-2R -chain and the lower affinity Fc receptor for
IgE (CD23/FceRII) that are now known to be under the control of
NF-B. Moreover, Schenk et al. (42) observed that TRX could augment
the tetradecanoyl phorbol acetate-induced production of IL-1, IL-2,
IL-8, and TNF- from four distinct cell lines including U937 and
Molt-4. Although they claimed that TRX could directly induce IL-6
production without any additional stimulus, we did not observe sole
effects of TRX in IL-6 production from RSFs using the extensively
purified TRX by polymyxin B to exclude LPS contamination. We also found
that TRX could augment the phosphorylation and thus degradation of
IB that was elicited by TNF- (Fig. 6). TNF is known to
initiate signaling through TNF receptor-associated signal transducer
factor 2 (50). TNF receptor-associated signal transducer factor 2
activates NF-B-inducing kinase (51), which then activates IB
kinase (IKK) complex consisting of IKK- and IKK-ß (52, 53).
We and others have previously demonstrated that the DNA-binding
activity of NF-B was stimulated by TRX (35, 36, 55, 56). Together
with these findings, we propose that TRX may stimulate the NF-B
signaling by two independent mechanisms: 1) facilitating the signaling
pathway leading to IB phosphorylation by IKK from outside of the
cell; and 2) increasing the NF-B DNA binding by direct contact as
previously demonstrated. Thus, TRX monitoring in patients with RA would
provide a useful information regarding the extent of oxidative stress.
Furthermore, the positive correlation between the SF TRX and the serum
CRP in the absence of a high concentration of SF TNF- may indicate
that TRX is involved in the prolongation and persistence of the
rheumatoid inflammation, because high concentrations of TRX could
stimulate NF-B activation in the presence of the otherwise
insufficient concentration of TNF-.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Takashi Okamoto, Department of Molecular Genetics, Nagoya City University Medical School, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan. E-mail address:
3 Abbreviations used in this paper: RA, rheumatoid arthritis; CRP, C-reactive protein; ROI, reactive oxygen intermediate; RSF, rheumatoid synovial fibroblast; SF, synovial fluid; TRX, thioredoxin; Hb, hemoglobin; RF, rheumatoid factor; IKK, IB kinase.
Received for publication January 26, 1999. Accepted for publication April 7, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) SF
TNF-
, 10; 
, 100; •, 500 ng/ml) were added to the cultured RSFs
(0.9 x 105 cells) 1 h before TNF-
