Keywords chondrocytes; extracellular signal-regulated kinase ERK; monocyte chemoattractant protein-4 MCP-4 ⁄ CCL13; rheumatoid arthritis Correspondence H.. Abbreviations DAB, 3¢3-diamino
Trang 1(MCP-4)/CCL13 from chondrocytes in rheumatoid arthritis Takuji Iwamoto1,2, Hiroshi Okamoto1, Shu Kobayashi1,2, Katsunori Ikari1, Yoshiaki Toyama2,
Taisuke Tomatsu1, Naoyuki Kamatani1and Shigeki Momohara1
1 Institute of Rheumatology, Tokyo Women’s Medical University, Japan
2 Department of Orthopedic Surgery, School of Medicine, Keio University, Tokyo, Japan
Rheumatoid arthritis (RA) is a chronic, symmetric
poly-articular joint disease that primarily affects the small
joints of the hands and feet [1] It is characterized by
infiltration of inflammatory cells such as monocytes
and T-lymphocytes into the joints, leading to synovial
proliferation and progressive destruction of cartilage
and bone [2] Although the basic mechanisms of RA
are widely accepted, the pathogenesis of the disease is
not fully understood
Chemokines in humans comprise more than 50 small
(8–10 kDa) heparin-binding proteins that were
origi-nally identified by their chemotactic activity on bone
marrow-derived cells [3,4] They are classified into
four families on the basis of the location of cysteine
residues The four chemokine groups are CC, C, CXC, and CX3C, and their receptors are consequently classi-fied as CCR, CR, CXCR, and CX3CR Chemokines and chemokine receptors have been shown to be involved in a variety of inflammatory diseases by recruiting leukocytes to the inflammatory site [5] It is well known that synovial tissue and synovial fluid from
RA patients contain increased concentrations of sev-eral chemokines, such as interleukin (IL)-8)⁄ CXCL8, interferon-c (IFN-c)-inducible protein-10⁄ CXCL10, monokine induced by interferon-c⁄ CXCL9, stromal cell-derived factor-1⁄ CXCL12, monocyte chemotactic protein (MCP)-1⁄ CCL2, macrophage inflammatory protein-1a⁄ CCL3, and fractalkine⁄ CXC3CL1 [6]
Keywords
chondrocytes; extracellular signal-regulated
kinase (ERK); monocyte chemoattractant
protein-4 (MCP-4) ⁄ CCL13; rheumatoid
arthritis
Correspondence
H Okamoto, Institute of Rheumatology,
Tokyo Women’s Medical University, 10-22
Kawada-cho, Shinjuku, Tokyo 162-0054,
Japan
Fax: +81 3 5269 1726
Tel: +81 3 5269 1725
E-mail: hokamoto@ior.twmu.ac.jp
(Received 14 April 2007, revised 6 July
2007, accepted 26 July 2007)
doi:10.1111/j.1742-4658.2007.06013.x
We studied the role of monocyte chemoattractant (MCP)-4⁄ CCL13 in the pathogenesis of rheumatoid arthritis (RA) MCP-4 was highly expressed in cartilage from RA patients Interferon-c significantly stimulated MCP-4⁄ CCL13 production in human chondrocytes, and this effect was enhanced in combination with interleukin-1b or tumor necrosis factor-a MCP-4⁄ CCL13 induces the phosphorylation of extracellular signal-regulated kinase
in fibroblast-like synoviocytes and activates cell proliferation, and PD98059 completely inhibits these effects These data suggest that interferon-c in combination with interleukin-1b⁄ tumor necrosis factor-a activates the pro-duction of MCP-4⁄ CCL13 from chondrocytes in RA joints, and that secreted MCP-4⁄ CCL13 enhances fibroblast-like synoviocyte proliferation
by activating the extracellular signal-regulated kinase mitogen-activated protein kinase cascade
Abbreviations
DAB, 3¢3-diaminobenzidine tetrahydrochloride; ERK, extracellular signal-regulated kinase; FLS, fibroblast-like synoviocyte; IFN-c, interferon-c;
IL, interleukin; MCP, monocyte chemoattractant protein; OA, osteoarthritis; RA, rheumatoid arthritis; SNP, single-nucleotide polymorphism; TNF-a, tumor necrosis factor-a; XTT, sodium 3¢-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate.
Trang 2These chemokines are implicated in RA pathogenesis
via the recruitment and retention of leukocytes into
the joints In addition to functioning in cell traffic,
several chemokines are reported to enhance the
prol-iferation of fibroblast-like synoviocytes (FLSs) and
upregulate gelatinase and collagenase production by
FLSs [7] Thus, chemokines are key molecules in RA
pathogenesis and are potential therapeutic targets for
RA [8]
Although macrophages and FLSs are considered to
be the most potent producers of chemokines in the
synovial compartment, chondrocytes also have the
ability to produce chemokines [8–10] In our previous
report, we found that mRNA expression of
MCP-4⁄ CCL13 was significantly higher in cartilage from RA
patients than from osteoarthritis (OA) patients or
nor-mal controls, and the concentration of MCP-4⁄ CCL13
protein in synovial fluid was also significantly higher in
RA patients than in OA patients [11]
MCP-4⁄ CCL13 is a recently identified CC
chemo-kine from a human cDNA library that directs the
migration of eosinophils, monocytes and
T-lympho-cytes through several chemokine receptors, including
CCR-2 and CCR-3 [12,13] The role of MCP-4⁄
CCL13 in disease is less well defined, but recent studies
suggest that it is involved in inflammatory cell
recruit-ment in allergic disorders such as asthma and atopic
dermatitis [14–17]
In the present study, we further determined the role
of MCP-4⁄ CCL13 in RA pathogenesis We
investi-gated the role of several stimuli on the expression of
MCP-4⁄ CCL13 by human chondrocytes, and the
sig-nal transduction pathways controlling FLS
prolifera-tion by MCP-4⁄ CCL13 In addiprolifera-tion, we conducted a
case-control study using single-nucleotide
polymor-phisms (SNPs) to determine whether MCP-4⁄ CCL13
could be a genetic risk factor for RA
Results
Production of MCP-4⁄ CCL13 by human
chondrocytes
To identify the stimulatory signals that activate the
production of MCP-4⁄ CCL13 from human
chondro-cytes, we investigated the effect of several cytokines
reported to have roles in RA pathogenesis Human
chondrocytes from RA or OA patients were cultured
in the presence of IL-1b, tumor necrosis factor-a
(TNF-a) or IFN-c, and various combinations of these
three cytokines Chondrocytes from both RA and
OA patients gave similar results, and we present the
data obtained with RA-derived chondrocytes
MCP-4⁄ CCL13 protein concentrations in culture superna-tants were evaluated by ELISA IFN-c significantly stimulated MCP-4⁄ CCL13 production in a dose-depen-dent manner, whereas IL-1b and TNF-a had no signif-icant effect (Fig 1A) Interestingly, stimulation of MCP-4⁄ CCL13 production by IFN-c was significantly and remarkably enhanced when IFN-c was combined with IL-1b or TNF-a (Fig 1B)
To determine whether these observed effects occur
at the transcriptional level, quantitative real-time PCR analysis was performed on IL-1b, TNF-a, and IFN-c Consistent with the ELISA data, IFN-c significantly stimulated mRNA expression of MCP-4⁄ CCL13 in
0 100 200 300
IFN-γγ
0 1 10 100 1000
400
A
B
0 10 100 0 10 100
*
*
0 100 200 300
IFN- γ (ng/mL)
400 500 600
IL-1 β (ng/mL) TNF- α (ng/mL)
10 10 10
100 100 10 100 10
#
#
1000
Fig 1 MCP-4 ⁄ CCL13 protein production by human chondrocytes from RA and OA patients Human chondrocytes were cultured in the presence of (A) IL-1b (0–100 ngÆmL)1; open bars), TNF-a (0–100 ngÆmL)1; solid bars) and IFN-c (0–1000 ngÆmL)1; shaded bars) for 48 h, or (B) IFN-c (shaded bars), IFN-c + IL-1b (open bars), and IFN-c + TNF-a (solid bars) for 48 h MCP-4 ⁄ CCL13 protein concentrations in these cell culture supernatants were evaluated by ELISA Bars show the mean and SD of four separate experiments Statistical evaluation was performed using one-way ANOVA followed by Tukey’s method for multiple comparisons *P < 0.01 compared with vehicle-treated control. #P < 0.01 compared with the sample cultured with IFN-c alone.
Trang 3a dose-dependent manner IL-1b or TNF-a also
enhanced the mRNA expression of MCP-4⁄ CCL13
induced by IFN-c Chondrocytes from both RA and
OA patients gave similar results, and we present the
data obtained with RA-derived chondrocytes These
results suggest that these stimulatory effects occur at
the transcriptional level (Fig 2A,B)
Effect of MCP-4⁄ CCL13 on extracellular
signal-regulated kinase (ERK) phosphorylation
Western blot analysis was performed to investigate
whether MCP-4⁄ CCL13 could activate ERK, which is
widely known to play a major role in cell
prolifera-tion [20] The FLSs from both RA and OA patients were similar, and we present the data from the RA-derived FLSs As expected, phosphorylation of ERK was induced by stimulation with MCP-4⁄ CCL13 in RA FLSs This activation peaked at about 10–20 min, and returned to basal levels within 60 min (Fig 3A) Incubation with 100 lm PD98059, a spe-cific ERK activation inhibitor, was sufficient to abol-ish ERK activation by MCP-4⁄ CCL13 in RA FLSs (Fig 3B)
To confirm the involvement of ERK phosphorylation
in the proliferative effect on RA FLSs of MCP-4⁄ CCL13, an XTT {sodium 3¢-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis-(4-methoxy-6-nitro) benzene sulfo-nic acid hydrate} cell proliferation assay was performed, using PD98059 to antagonize the phosphorylation of ERK As representative results of western blot ana-lyses were obtained at a concentration of 100 ngÆmL)1,
an XTT cell proliferation assay was performed with the same concentrations MCP-4⁄ CCL13 enhanced the proliferation of FLSs in a dose-dependent manner, and PD98059 completely inhibited the stimulatory effect of MCP-4⁄ CCL13 on FLS proliferation PD98059 did not inhibit basal proliferation of RA FLSs (Fig 4A) The FLSs from both RA and OA patients were similar, and we present the data from the RA-derived FLSs
Association study using SNPs of MCP-4⁄ CCL13
As we found that MCP-4⁄ CCL13 was one of the key molecules in the pathogenesis of RA, we studied the association of the MCP-4⁄ CCL13 gene with RA sus-ceptibility We selected two SNPs in MCP-4⁄ CCL13 (T887C and rs159313), for the following reasons Two single-nucleotide T–to–C polymorphisms (T896C and T887C) were reported in the MCP-4⁄ CCL13 core pro-moter region [22] They are located 896 and 887 bp before the transcription initiation site, and were reported to have direct effects on the transcript level of the gene [22] After preliminary analysis, we selected T887C for a larger-scale study, because T896C and T887C were in complete linkage disequilibrium (D¢ ¼ 1.0, r2¼ 1.0) On the basis of information from the National Center of Biotechnology Information data-base, three SNPs (rs3136677, rs159313 and rs2072069) were found in MCP-4⁄ CCL13 Among them, we selected SNP rs159313 for the study, because one SNP (rs3136677) was nonpolymorphic in Japanese popula-tions, and other two SNPs (rs159313 and rs2072069) were in complete linkage disequilibrium (D¢ ¼ 1.0,
r2¼ 1.0) according to the International HapMap pro-ject (public release 19) [23]
IFN-γ
IFN-γ 1000 ng/mL IFN-γ 100 ng/ml IFN-γ 10 ng/ml
1 ng/ml IFN-γ 0.1 ng/ml
IFN-γ 1000 IFN-γ 100 ng/mL IFN-γ 10 ng/mL
1 ng/mL IFN-γ 0.1 ng/mL
Time (hours)
*
*
2
4
6
8
A
B
IFN-γ 10 ng/mL + Il -1β 10 ng/mL
IFN-γ + IL-1β IFN-γ 10 ng/mL
Il -1β 10 ng/mL + TNF-α 10 ng/mL IL-1β + TNF-α TNF-α 10 ng/mL TNF-α
Il -1β 10 ng/mL IL-1β
IFN-γ 10 ng/mL + TNF-α 10 ng/mL
IFN-γ + TNF-α
2
4
0
6
8
Time (hours)
# #
Fig 2 MCP-4 ⁄ CCL13 mRNA expression by human chondrocytes
from RA and OA patients Total RNA from chondrocytes stimulated
with different cytokines for 4–12 h was harvested and transcribed
to cDNA by reverse transcription cDNA was used for TaqMan
quantitative real-time PCR: (A) with IFN-c (0–1000 ngÆmL)1), and (B)
with IL-1b, TNF-a, IFN-c, IL-1b + TNF-a, IFN-c + IL-1b, and
IFN-c + TNF-a The figure shows expression of MCP-4 ⁄ CCL13
mRNA relative to time-matched vehicle-treated controls using the
comparative threshold cycle (Ct) method Data are mean ± SD of
four separate experiments Statistical evaluation was performed
using one-way ANOVA followed by Tukey’s method for multiple
comparisons *P < 0.01 compared with vehicle-treated control.
# P < 0.01 compared with the sample cultured with IFN-c alone.
Trang 4According to the genotyping results, these two SNPs
were present in Hardy–Weinberg equilibrium in both
cases and controls No statistically significant
differ-ences in genotype or allele frequencies were observed
between cases and controls We failed to find
signifi-cant differences even when RA patients were stratified
according to the rheumatoid factor status (Table 1)
These data indicate that the MCP-4⁄ CCL13 gene may
not be responsible for the onset of RA
Discussion
MCP-1⁄ CCL2, MCP-2⁄ CCL8, MCP-3⁄ CCL7 and
MCP-4⁄ CCL13 constitute a subfamily of CC
chemo-kines that share structural and functional features
MCP-1⁄ CCL2 was the first to be identified [24], and
MCP-4⁄ CCL13 is the most recently identified
chemo-kine, and is a potent chemoattractant for eosinophils,
monocytes and T-lymphocytes [12,13] MCP-4⁄ CCL13
expression is upregulated at sites of inflammation in a
number of different diseases, including asthma [14–16],
atherosclerosis [25], acute renal inflammation [26], and
atopic dermatitis [17] MCP-4⁄ CCL13 was also highly
expressed in articular cartilage from patients with RA
[11]
In the present study we demonstrated
MCP-4⁄ CCL13 protein production by human chondrocytes,
and showed for the first time that MCP-4⁄ CCL13
from chondrocytes is actively involved in RA
patho-genesis We also demonstrated that IFN-c was the
main stimulus for MCP-4⁄ CCL13 production, and that TNF-a and IL-1b enhanced the stimulatory effect of IFN-c According to the analysis of the human
MCP-4⁄ CCL13 gene in dermal fibroblasts, the core promoter region contained IFN-c-response elements as well as nuclear factor-jB-like consensus sequences [27] Fur-thermore, MCP-4⁄ CCL13 mRNA expression was reported to be upregulated by stimulation with TNF-a and IFN-c in dermal fibroblasts [27] Similar results were obtained in human airway epithelial cell lines after stimulation with the cytokine TNF-a alone or in combination with IFN-c [28] In contrast, our experi-ments indicated that IFN-c was the main stimulus for MCP-4⁄ CCL13 production by human chondrocytes
We observed no significant stimulation of induction of MCP-4⁄ CCL13 mRNA expression by TNF-a alone in human chondrocytes, whereas TNF-a greatly enhanced the expression in combination with IFN-c
IFN-c is produced by T-cells and by natural killer cells infiltrating the inflamed synovium, and is secreted into the joint space, although its role in the progres-sion of articular injury remains controversial [29] To date, divergent in vitro effects of IFN-c have been reported in the literature IFN-c induces the produc-tion of nitric oxide, IL-6 and prostaglandin E2 by human chondrocytes [30] In contrast, IFN-c inhibits TNF-a- and IL-1b-induced collagenase and stromely-sin production by chondrocytes, as well as TNF-a-and IL-1b-stimulated proteoglycan degradation [31,32] Furthermore, the effects of IFN-c in the treatment of
pERK
A
B
1/2
ERK 1/2
Relative Intensity 1.00 1.03 16.55 47.73 46.16 23.29 1.04 1.05
Relative Intensity 1.00 0.96 0.94 0.95 0.98 0.97 0.99 1.01
pERK 1/2
ERK 1/2
Relative Intensity 1.00 40.01 9.20 0.96 27.88
Relative Intensity 1.00 0.96 0.98 1.01 1.03
Fig 3 Induction of ERK phosphorylation by
MCP-4 ⁄ CCL13 in RA FLSs FLSs were
cul-tured overnight in serum-free DMEM (A)
FLSs were incubated in the presence of
MCP-4 ⁄ CCL13 (100 ngÆmL)1) for an
addi-tional 0–120 min (B) FLSs were incubated
in the presence of MCP-4 ⁄ CCL13
(100 ngÆmL)1) for 20 min with or without
PD98059 (10–100 lgÆmL)1) As a positive
control, FLSs were incubated with IL-1b
(5 ngÆmL)1) for 20 min Cell lysates were
examined for ERK activation by western
blotting with phospho-p44 ⁄ 42 MAP kinase
mouse monoclonal antibody (pERK1 ⁄ 2).
Total p44 ⁄ 42 MAP kinase antibody was
used to verify equal protein loading The
result is one representative example from
three independent experiments.
Trang 5RA are unclear A statistically significant improvement
was observed among the RA patients treated with
recombinant IFN-c in one double-blind study of 91
patients [33], whereas current evidence shows that
anti-IFN-c therapy is significantly superior to placebo in 30
patients with RA [34] It is widely accepted that
TNF-a is the key molecule in RA pathogenesis, as
demonstrated by the clinical benefit of
TNF-a-neutra-lizing therapy [35] Although approximately 40% of
patients show dramatic responses, the remainder show some evidence of persistent synovitis or minimal clini-cal benefit [1] The results of the present study suggest that IFN-c may contribute to the progression of joint inflammation, in part by modulating MCP-4⁄ CCL13 production by human chondrocytes
We have also demonstrated the ability of
MCP-4⁄ CCL13 to phosphorylate ERK mitogen-activated protein (MAP) kinase, and have shown that induction
of FLS proliferation by MCP-4⁄ CCL13 is dependent
on the phosphorylation of ERK MAP kinase It is widely accepted that the progressive destruction of articular cartilage is reliant on the evolution of hyper-plastic synovial tissue, and that hyperplasia of FLSs is dependent on dysregulated proliferation and apoptosis [1,36] Key regulators of this proliferation include the recently recognized macrophage migration inhibitory factor and proinflammatory cytokines such as TNF-a and IL-1b through the nuclear factor-jB and⁄ or MAP kinase signal transduction pathways [37–40] To date, several chemokines, including MCP-1, stromal cell-derived factorF-1a, IFN-c-inducible protein, monokine induced by IFN-c and MCP-4⁄ CCL13 are also known
to enhance FLS proliferation, although the signal transduction pathways underlying the proliferation remain unclear [7,11] We hypothesized that ERK acti-vation might be involved in the proliferative effect
of MCP-4⁄ CCL13, as the ERK cascade has been reported to be a central pathway that transmits signals from many extracellular agents to regulate cellular pro-cesses such as proliferation, differentiation and cell cycle progression in various cells [22,41] As expected, the MCP-4⁄ CCL13–ERK cascade was indeed involved
in the proliferation of synovial cells, as shown here In addition, ERK is reported to have a role in the expres-sion of matrix metalloproteinases (MMPs), such as MMP-1, MMP-2, and MMP-9, and contributes to the degradation of extracellular matrix for the invasion of melanoma cells [42] Several lines of evidence have shown that MMPs are involved in the joint degrada-tion process in RA Thus, MCP-4⁄ CCL13 might have roles not only in the proliferation of synovial cells but also in the invasion of synovial cells, resulting in pan-nus formation and destruction of joints in RA Taken together with these results, MCP-4⁄ CCL13 secreted from chondrocytes in the joints plays an important role in the development of aggressive synovial tissues
in RA, as illustrated in Fig 4B There are numerous reports showing the importance of synovial cells in RA pathogenesis Our data support the notion that chon-drocytes are also actively involved in RA pathogenesis
In conclusion, we have shown that MCP-4⁄ CCL13
is produced by human chondrocytes from RA patients
0.8
0.9
1.0
1.1
MCP-4 (ng/mL)
PD98059
*
**
*
10 0 +
+
100 10 +
**
1.2
A
MCP-4
Proliferation
IL-1β, TNF-α
Synovial Cells
IL-18
Fig 4 (A) Effects of inhibition of ERK phosphorylation on RA
FLS proliferation FLSs were treated with MCP-4 ⁄ CCL13 (0–
100 ngÆmL)1) with and without the addition of MAP kinase kinase
inhibitor PD98059 (100 lgÆmL)1) for 48 h MCP-4 ⁄ CCL13
signifi-cantly increased RA FLS proliferation, and inhibition of ERK
phos-phorylation by PD98059 significantly inhibited FLS proliferation.
PD98059 did not inhibit basal proliferation of RA FLSs Bars show
the mean and SD of three indepemdent experiments Statistical
evaluation was performed using one-way ANOVA followed by
Tukey’s method for multiple comparisons.*P < 0.05; **P < 0.01.
(B) Schematic representation of the role of MCP-4 ⁄ CCL13 in RA A
vicious circle is formed between chondrocytes and synovium in the
affected joint IFN-c is produced by Th1 (T helper 1) cells infiltrating
the synovium and activates the expression of MCP-4 ⁄ CCL13 Then,
MCP-4 ⁄ CCL13 stimulates the proliferation of synovial cells, which
produce inflammatory cytokines (IL-b, TNF-a) Together, these
cyto-kines, with IFN-c, further enhance the production of MCP-4 ⁄ CCL13
by chondrocytes.
Trang 6stimulated by IFN-c and TNF-a⁄ IL-1b In addition,
MCP-4⁄ CCL13 has significant effects on FLS
prolifer-ation that are dependent on the activprolifer-ation of ERK
MAP kinase These data suggest that MCP-4⁄ CCL13
is a significant contributor to synovial hyperplasia in
RA, and that MCP-4⁄ CCL13 may serve as a new
tar-get for anti-RA therapy
Experimental procedures
Preparation of articular cartilage and synovial
tissue
Human articular cartilage and synovial tissue were obtained
from OA and RA patients (n¼ 5 in each group) who were
undergoing total knee replacement at Tokyo Women’s
Medical University, Tokyo, Japan OA was diagnosed by
physical examination along with radiographic findings, and
RA patients met the 1987 disease criteria of the American
College of Rheumatology [18] All samples were obtained
with informed consent All experiments were approved
by the Ethical Committee of Tokyo Women’s Medical
University
Isolation and culture of chondrocytes and FLSs
Tissue was obtained under aseptic conditions and was finely
minced Chondrocytes were isolated by sequential
enzy-matic digestion at 37C: 5 mgÆmL)1pronase (Kaken
Phar-maceutical Co., Ltd, Tokyo, Japan) for 1 h, followed by
2 mgÆmL)1 collagenase (Sigma Chemical Co., St Louis,
MO, USA) for 6 h at 37C in DMEM (Nikken Bio
Medi-cal Laboratory, Kyoto, Japan) with antibiotics (100 unitsÆ
mL)1 penicillin, 100 lgÆmL)1 streptomycin; Gibco BRL,
Grand Island, NY, USA) FLSs were also isolated by
diges-tion with 1 mgÆmL)1 collagenase for 3 h at 37C in
DMEM The digested tissue was briefly subjected to centri-fugation at 1500 g at 37C for 15 min using an MX-100 centrifuge (TOMY Seiko, Tokyo, Japan) with TMP-11 angle-type rotor, and the resulting pellet was washed three times in NaCl⁄ Pi The isolated cells were seeded at high density in tissue culture flasks and cultured in DMEM sup-plemented with 10% heat-inactivated fetal bovine serum (Tissue Culture Biologicals, Tulare, CA, USA) at 37C in
a humidified atmosphere of 5% CO2⁄ 95% air The culture medium was changed every 3–5 days, and nonadherent lymphoid cells were removed At confluence, chondrocytes and FLSs were detached and passaged once, and then seeded at high density and allowed to grow in DMEM supplemented as above Chondrocytes were used between passages 1 and 3, and FLSs were used between passages
5 and 8 for the following experiments In some cases, carti-lage tissue slices were obtained for immunohistochemical analysis
Effect of cytokines on MCP-4 production by human chondrocytes
Human chondrocytes from four RA patients and three OA patient were cultured in DMEM supplemented with 10% fetal bovine serum in 12-well culture plates At confluence, the culture medium was replaced with serum-free DMEM After 24 h, chondrocytes were incubated for an additional
48 h in the absence or presence of recombinant human IL-1b (0–100 ngÆmL)1; R&D Systems), recombinant human TNF-a (0–100 ngÆmL)1; R&D Systems), recombinant human IFN-c (0–1000 ngÆmL)1; R&D Systems) and combi-nations of these cytokines The culture supernatant was collected and stored at ) 80 C MCP-4 concentrations in these supernatants were evaluated as described above Experiments were performed three times with each of the four independent cultures
Table 1 Summary of the association of MCP-4 in rheumatoid arthritis cases and controls The major allele was always referred to as allele 1 and the minor allele as allele 2 SNP, single-nucleotide polymorphism; RF, rheumatoid factor; MAF, minor allele frequency; OR, odds ratio; 95% CI, confidence interval.
SNP
Genotype
rs159313
T-887C
a Distribution of the frequency of allele 1 versus allele 2 in the cases compared with the controls.
Trang 7Quantitative real-time PCR
Total RNA was harvested from chondrocytes stimulated
with cytokines for 4–12 h using the RNeasy Mini Kit
according to the manufacturer’s instructions (Qiagen,
Chats-worth, CA, USA) cDNA was synthesized from 0.3 lg of
total RNA in a 20 lL reaction using TaqMan Reverse
Tran-scription Reagents (Applied Biosystems, Tokyo, Japan)
TaqMan quantitative real-time PCR was performed using
the ABI Prism 7900HT sequence detection system and
Taq-Man PCR Master Mix according to the manufacturer’s
pro-tocol (Applied Biosystems) Primers and probes for human
MCP-4⁄ CCL13 and human glyceraldehyde-3-phosphate
dehydrogenase were purchased from Applied Biosystems
RNA samples lacking reverse transcriptase were used with
each real-time PCR experiment to verify the absence of
genomic DNA The incubation was initiated at 50C for
2 min, and this was followed by 95C for 10 min, and 40
cycles at 95C for 15 s and 65 C for 1 min Samples were
compared using the comparative threshold cycle (Ct)
method to determine MCP-4 mRNA expression relative to
the time-matched vehicle-treated control The parameter Ct
is the PCR cycle number at which the fluorescence generated
by cleavage of the probe reaches a fixed threshold above
baseline For each sample, the MCP-4⁄ CCL13 Ct value was
normalized using DCt¼ MCP-4 ⁄ CCL13 Ct)
glyceralde-hyde-3-phosphate dehydrogenase Ct To determine relative
expression levels, the following formula was used: DDCt¼
sample DCt) time-matched control DCt, and the value used
to plot relative MCP-4⁄ CCL13 expression of each sample
was calculated using the expression 2–DDCt
Western blot analysis
The phosphorylation of p44⁄ 42 MAP kinase, or ERK, was
assessed by western blotting In brief, FLSs were cultured in
DMEM supplemented with 10% fetal bovine serum on a
10 cm culture dish At 80% confluence, the culture medium
was replaced with serum-free DMEM After 24 h, FLSs
were incubated in the presence of recombinant human
MCP-4 (rHuMCP-4, 100 ngÆmL)1; R&D Systems) for an
additional 0–120 min In addition, FLSs were incubated in
the presence of recombinant human MCP-4 (100 ngÆmL)1)
for 20 min with or without a specific inhibitor of MAP
kinase kinase, PD98059 (Calbiochem, San Diego, CA, USA;
10–100 lgÆmL)1) As a positive control, FLSs were
incu-bated with recombinant human IL-1b (5 ngÆmL)1) for
20 min Cells were lysed with Cell Lysis Buffer (Cell
Signal-ing Technology, Beverly, MA, USA) After incubation on
ice for 10 min, the protein concentration was determined,
and the lysates were stored at ) 80 C Equal amounts of
cellular proteins were separated by SDS⁄ PAGE and
trans-ferred to Immune-Blot poly(vinylidene difluoride)
mem-brane (Bio-Rad, Hercules, CA, USA) Immunoblotting was
performed using phospho-p44⁄ 42 MAP kinase mouse
monoclonal antibody (Cell Signaling Technology; diluted
1 : 5000) and p44⁄ 42 MAP kinase antibody (Cell Signaling Technology; diluted 1 : 1000) to verify equal protein loading
Cell proliferation assay FLSs were seeded at a density of 1· 103cells per well in 96-well microtiter plates in 100 lL of serum-free DMEM per well, and were treated with recombinant human
MCP-4 (0–100 ngÆmL)1) for 48 h The activation of ERK was antagonized with PD98059 (100 lgÆmL)1) Cell prolifera-tion was evaluated by measuring the number of viable cells using the XTT assay by using the XTT Cell Proli-feration Kit II (Roche Applied Science, Mannheim, Germany) [19] Formazan product in the supernatant was measured in terms of absorbance values at 490 nm by using an ELISA plate reader The absorbance values obtained from culture medium without cells were sub-tracted from the values obtained with cells Experiments were performed six times with each of the three indepen-dent cultures
Genetic association study using SNPs The study was part of an RA cohort project (IORRA: Institute of Rheumatology RA cohort), and was approved
by Tokyo Women’s Medical University Genome Ethics Committee [20] Out of the registered RA patients, DNA samples were obtained from 1284 Informed written consent was obtained from every subject Of these, 1128 samples were randomly selected for this study Eighty-eight per cent
of them were rheumatoid factor positive They were mostly females (82.6%), and the mean age of the patients was 57.6 years (range: 19–85 years) Four hundred and fifty-five population-based control DNA samples were obtained from the Pharma SNP consortium (http://www.jpma.or.jp/ psc/index.html) All control subjects were matched for sex, ethnic origin, and geographical area
SNP genotyping was performed using the TaqMan fluoro-genic 5¢-nuclease assay (Applied Biosystems) according to the manufacturer’s instructions, as described previously [21]
Statistical methods Data are presented as the mean ± standard deviation (SD) Statistical comparisons were performed using either the Mann–Whitney U-test or one-way ANOVA followed
by Tukey’s method for multiple comparisons, as appropri-ate Hardy–Weinberg equilibrium and associations between
RA and each of the SNPs were estimated by the chi-square test Statistical significance was established at the P < 0.05 level All analyses were carried out using the r software package, version 2.0.1 (http://www.r-project.org/)
Trang 8This work was supported, in part, by grants-in-aid
from the Ministry of Education, Culture, Sports,
Sci-ence and Technology of Japan The expert technical
help of Yukiko Katagiri is gratefully acknowledged
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