Yonago Acta medica 2013;56:13–19

Original Article

Etanercept Promotes Bone Formation via Suppression of Dickkopf-1 Expression in
Rats with Collagen-Induced Arthritis
Atsushi Tanida,* Yuji Kishimoto,* Toru Okano* and Hiroshi Hagino†*Division of Orthopedic Surgery, Department of Medicine of Sensory and Motor Organs, School of Medicine, Tottori University Facultyof Medicine, Yonago 683-8504, Japan and †Department of Fundamental Nursing, School of Health Science, Tottori University Facultyof Medicine, Yonago 683-8503, Japan

tive, independent of the regulation of osteoclast activity.
Marked bone formation is attributed to the fact that ETN
directly promotes osteoblastogenesis, not as a result ofsuppressing osteoclastogenesis.

ABSTRACT
Background

Various clinical reports suggest etanercept (ETN) has some efficacy in bone formation inrheumatoid arthritis (RA). To examine this effect, weinvestigated the gene expression of cytokines relevant toosteoblast/osteoclast differentiation, and evaluated histomorphometric findings in mature rats with collageninduced arthritis (CIA).
Methods Total RNA was extracted from knee joints
with CIA after ETN or placebo administration. Subsequently, realtime-PCR was carried out to quantify themRNAs encoding Wnt-1, Dickkopf-1 (DKK-1), receptor
activator of nuclear factor kappa-B ligand (RANKL),
osteoprotegelin (OPG) and TNF (tumor necrosis factor)alpha. In histomorphometric analysis, the infiltrating
pannus volume and pannus surface, and the following
items in contact with pannus surface were measured: osteoclast number, osteoid surface, osteoid volume and labeling surface. These were evaluated in the distal femur
with CIA with or without ETN administration.
Results
TNF-alpha, RANKL and OPG mRNA
expressions, linked to osteoclastogenesis, were notsignificantly different with or without ETN administration. ETN administration significantly increased Wnt-1
mRNA expression, the osteoblast promoter, and decreased DKK-1 mRNA expression, the Wnt signal inhibitor. In histomorphometric analysis, pannus volume,
pannus surface and osteoclast number, parameters ofbone destruction, were not significantly different amonggroups. Osteoid volume, osteoid surface and labelingsurface, parameters of bone formation, increased significantly with ETN administration.
Conclusion Our results suggest that ETN suppresses
DDK-1 expression, and, as a result, Wnt expression is
promoted and osteoblastogenesis becomes more ac-

Key words

bone formation; Dickkopf-1; etanercept;
rheumatoid arthritis; Wnt

Rheumatoid arthritis (RA) is a chronic inflammatory
disease, accompanied by synovitis, that causes joint destruction. Various cytokines, including tumor necrosis
factor (TNF)-alpha, have been implicated as important
mediators of inflammation and joint destruction in
RA.1–3 The scientific consensus is that the receptor activator of nuclear factor kappa-B ligand (RANKL) andosteoprotegelin (OPG) system is pivotal to the pathogenesis of bone erosions in RA.4 Also, TNF-alpha promotes
the expression of RANKL, and that is associated with
up-regulation of osteoclast activity.5
From the point of view of bone metabolism, it has
also become clear that TNF-alpha regulates the activity of osteoblasts, which play a role in bone metabolism
with osteoclasts.6–8 Recently, it was reported that Dickkopf-1 (DKK-1), an inhibitor of the Wnt signaling pathway, regulated the balance between osteoclastogenesis
and osteoblastogenesis in an inflammatory joint RA
model. TNF-alpha may suppress osteoblast differentiation via promoting the expression of DKK-1, inhibiting
Wnt the signaling pathway.9–11
In recent years, the development of TNF-alphatargeted biologic agents has provided an excellent curative effect.12–15 As previously reported, a number ofclinical reports have described radiographic evidence ofthe repair of erosion in RA.16–19 The administration ofetanercept (ETN), an anti TNF-alpha agent, reduced thetotal Sharp score from the baseline established before
the initiation of therapy in a large clinical study.15 ETN
may promote bone formation, as well as inhibiting bone
destruction via suppression of TNF-alpha function. Although there are some indications that destructive bonelesions can be repaired in RA joints, the mechanism of
this repair remains unknown and histologically unproven. To examine the effects of ETN on bone formation,

Corresponding author: Hiroshi Hagino, MD
hagino@med.tottori-u.ac.jp
Received 2012 November 30
Accepted 2012 December 19
Abbreviations: CIA, collagen-induced arthritis; DKK-1, Dickkopf-1;
ETN, etanercept; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HPF, high power field; OPG, osteoprotegelin; RA, rheumatoid arthritis; RANKL, receptor activator of nuclear factor kappa-B
ligand; TNF, tumor necrosis factor

13

A. Tanida et al.

Tissue collection and RNA isolation
After the animals were killed, the bilateral distal femur
and proximal tibia (cut off 5 mm from the joint line) werecollected and cooled with liquid nitrogen immediately.
The bone tissues were collected aseptically and all connective tissue, including the periosteum, was completely
removed. Frozen bones were homogenized in appropriate
volumes in an Isogen Kit (Nippon Gene, Tokyo) using
POLYTRON (KINEMATICA AG, Tokyo). Total RNA
was extracted from the tissue homogenates according to
the manufacturer’s instructions. Additionally, we defecated the extracted total RNA using an RNeasy plus minikit (Quiagen, Hilden, Germany). RNA concentrations
were determined by spectrophotometry.

we i) investigated the gene expression of cytokines relevant to osteoblast differentiation within bone infiltrated
by pannus (Experiment A), and ii) performed histological evaluation of bone repair (Experiment B) in maturerats with collagen-induced arthritis (CIA), an animal
experimental model of RA.20
MATERIALS AND METHODS
Animals

Seven-month-old female Sprague-Dawley rats (retired
breeding animals, body weight 266–381 g, Shimizu
Laboratory Supply, Kyoto, Japan) were used in thisexperiment. This experiment was carried out in accordance with the Guidelines for Animal Experimentationof the Faculty of Medicine, Tottori University. During
this experiment, animals were freely fed with tap waterand food (CE-2; CLEA, Tokyo, Japan; calcium content
1.18 g/100 g, phosphorus content 1.09 g/100 g, vitamin
D3 content 250 IU/100 g). The room temperature was
set to 24 ˚C, and after about 2 weeks of preliminary
breeding, the animals were used for the experiments.

Reverse transcription
The total RNA was reverse-transcribed into cDNA using
M-MLV Reverse Transcriptase (Invitrogen, Carlsbad,
CA). The conditions were as follows: incubation 60 min
at 37 ˚C, reaction inactivation by heating at 95 ˚C for 5min, and maintenance at 4 ˚C.
Real-time PCRRat primers were used to analyze DKK-1, Wnt-1,
RANKL, OPG, TNF-alpha and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, housekeeping gene),
respectively. Specific primers designed with reference
to the Universal ProbeLibrary Assay Design Center andUniversal ProbeLibrary Probe (Roche Applied Science,
Madison, WI) were used as shown in Table 1. A quantitative real-time PCR was performed to compare expression levels of each mRNA. EXPRESS qPCR Supermix
with Premixed ROX (Invitrogen) was used, with an
initial denaturation step of 20 s at 95 ˚C, followed by 45
cycles of 1 s at 95 ˚C, and 20 s at 60 ˚C as the annealing
temperature. Relative quantification of mRNA expression was calculated by a 7900HT Fast Real-Time PCR
System with SDS2.3 software (Applied Biosystems,
Carsbad, CA).

Collagen arthritis models

According to the method described by Trentham et al.,20
1 mL of emulsion containing 0.5 mg bovine type II collagen (0.3% acetic acid solution; K-41 Cosmo-Bio, Tokyo) and 0.5 mg of incomplete Freund’s adjuvant (52100021; Difco Laboratories, Detroit, MI) were injected
intracutaneously at three sites on the back of CIA grouprats, and 1 week later, a half dosage of the same emulsions was injected. In the non-immunized group (Ngroup), physiological saline was injected in the samemanner. The development of arthritis was judged by theredness and swelling in the ankle by same observer. The
onset of arthritis was observed about 2 weeks after immunization, and arthritis became severe until 4 weeks.
Administration of biologic agentsIn the ETN administration group, rats were subcutaneously administered 3 mg/kg ETN 3 times per week (Egroup). In the placebo-administered group, physiologicalsaline was injected in the same manner (P group).

Experiment B: Histological findings

Animal grouping and ETN administration
Twenty rats were immunized and divided into the Egroup (n = 10) and P group (n = 10), and killed 8 weeks
after CIA immunization. ETN administration was started 3 weeks after immunization because bone destruction
was observed histologically 3 weeks after immunization.21

Experiment A: Analysis of mRNA expression

Animal grouping and ETN administration
The animals were divided randomly into 3 groups: Egroup (n = 21), P group (n = 21) and non-immunizedgroup (N group) (n = 5). Administrations of ETN (Egroup) and placebo (P group) were started 1 day after
immunization, and 7 animals were killed 7, 14 and 21days after immunization in each group.

Preparation of hard tissue specimens
Histological findings in hard tissue specimens weretaken from the medial condyle of the femur in the midsagittal plane then compared between the groups. Bone
14

ETN promotes bone formation
Table 1. Primers used for real-time PCR
Molecule
TNF-alpha
RANKL
OPG
TRAP
DKK-1
Wnt-1
GAPDH

Left primer
Right primer

Used probe
numberProduct(base pairs)

Accession
number5’-agttggggagggagacctt-3’5’-catccacccaaggatgtttag-3’
5’-agacacagaagcactacctgactc-3’
5’-ggccccacaatgtgttgta-3’5’-gaagcaactcaaaattgtggaa-3’
5’-tcagccaattcggtataatcttg-3’
5’-gcctcttgcgtcctctatga-3’
5’-agcaccatccacgtatcca-3’5’-cgggaattactgcaaaaacg-3’5’-caatgatgccttcctcgatt-3’
5’-tcggcaagatcgtcaacc-3’
5’-gtagtcgcaggtgcaggatt-3’
5’-acaactttggcatcgtgga-3’
5’-cttctgagtggcagtgatgg-3’

71

60

L00981

2

90

AF187319

98

94

U9433065

60

M7611076

83

NM001106350

15

128

NM001105714

114

62

NM017008

DKK-1, Dickkopf-1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; OPG, osteoprotegelin; RANKL, receptor activator of nuclear
factor kappa-B ligand; TNF, tumor necrosis factor; TRAP, tartrate-resistant acid phosphatase.

tioned in the sagittal plane at a thickness of 5 μm with amicrotome (Leica Biosystems, Solms, Germany).

labeling by subctaneous injection of calcein at 10 mg/kg
was performed twice at 17 days and 1 day before the animals were killed, and tetracycline at 20 mg/kg was given once at 9 days before the animals were killed [schedule
1/7/1/7/1 (number of days of the 1st labeling/number ofdays between 1st and 2nd labeling/number of days of the
2nd labeling/number of days between 2nd labeling and3rd labeling/number of days between 3rd labeling andkilling)]. The labeling schedule was decided as the resultof trial and error of preliminary experiment. The knee
joint with larger swelling in each rat was collected withthe joint capsule and synovial membranes preserved,
and fixed in 70% alcohol. The remaining whole knee
joint was stained with Villaneuva bone stain for 7 days,
dehydrated in graded concentrations of ethanol, and embedded in methyl-methacrylate without decalcification.
The resulting blocks of knee joint specimens were sec-

Bone histomorphometry
Bone histomorphometric measurements of the femur
were made using a semiautomatic image analyzing system (System Supply, Nagano, Japan) and a fluorescent
microscope (eclipse 80i; Nikon, Tokyo) set at a magnification power of 400. Because it was difficult to quantify bone formation after definite destructive action, we
regarded osteoid production and bone calcification seenon the surface of the pannus as evidence of the repair oferosion. Twenty sections closest to the mid-sagittal planeof the medial femoral condyle were sampled for eachgroup, 2 sections per rat, each parameter was measured
and the average was compared among the different
groups. A line connecting the intersection of the epiphyFig. 1. Schematic presentations of unit
area (a) and infiltrating pannus (b).
a: Epiphyseal bone marrow is divided
into 3 regions (A1-A3) by 2 lines
connecting 2 points, each equallydividing lines E1-E2 and C1-C2
into 3 portions.
b: We defined the shaded area as thepannus infiltrating the epiphyseal
medulla of the posteromedial femoral condyle.
C, cartilage;
C-CL, the line connecting theremaining edge of theposterior cortex to theposterior cartilage;
EL, epiphyseal line;
FC, femoral condyle;PC, posterior cortex.

15

A. Tanida et al.

Fig. 2. Relative quantification of mRNA expression in bone. mRNA expressions of tumor necrosis factor (TNF)-alpha, receptor activatorof nuclear factor kappa-B ligand (RANKL) and osteoprotegelin (OPG) do not change with etanercept (ETN) administration. Treatment
with ETN tends to reduce the RANKL/OPG ratio. DKK-1 mRNA expression in the E group is lower than the P group. Wnt-1 mRNA expression is increased with ETN administration. N, non-immunized group; d, days after immunization. Values are the mean ± SD (n = 7).
*P < 0.05 versus the N group. †P < 0.05 versus the P group.

Statistical analysis
Statistical significance was analysed using the software package Dr. SPSS 2 for Windows 11.0.1J (SPSS,Tokyo). In experiment A, Fisher’s protected least significant difference procedure was performed after repeated-measures analysis of variance. In experiment B,
the Mann-Whitney U test was used to compare medianvalues. P < 0.05 was considered significant in both experiments.

seal line and the anterior cortex (E1) to the intersectionof the epiphyseal line and the posterior cortex (E2) was
equally divided into 3 portions, and the 2 division points
were connected to 2 points equally dividing into 3 theline connecting the anterior end (C1) with the posteriorend (C2) of the articular cartilage; the bone marrow wasthus divided into 3 regions: A1 to A3. We defined theA3 region as a unit area (tissue volume) (Fig. 1A).21 To
measure the pannus volume and pannus surface infiltrating the epiphyseal medulla of the posteromedial femoral
condyle, we chose the line connecting the remainingedge of the posterior cortex to the posterior cartilage(C-CL) (Fig. 1B). We defined the shaded area as the infiltrating pannus. The pannus surface was measured inthis area exclusive of the surface on the C-CL. Then, thefollowing items in contact with the pannus surface were
measured: osteoclast number, osteoid surface, osteoid
volume and labeling surface. We also counted the number of plasma cells, macrophages, and synovial bloodvessels per high power field (HPF) in the same area toassess the activity of the infiltrating pannus. The field
was inclusive of the pannus close to the bone surface,but exclusive of bone tissue.

RESULTS
Experiment A: Analysis of mRNA expression

Expression of TNF-alpha increased significantly withtime in the E group (P = 0.005), and tended to increaseover the course of time in the P group (P = 0.065) (Fig. 2).
Compared with the N group, significant differenceswere shown on day 21 in both the E and P groups (P =0.033, 0.035, respectively). Expression of RANKL in the
P group tended to increase over time, whereas in the Egroup it showed no marked change. In all groups and at
all times, there were no significant differences comparedto the N group. OPG mRNA expression on day 7 tended
16

ETN promotes bone formation
Table 2. Bone histomorphometry of the posteromedial femoral condyle in the mid-sagittal plane
Group

PV/TV
(%)

PS/TV
(µm/µm2)

Oc.N/PS
(/mm)

OV/PV
(%)

OS/PS
(%)

LS/PS
(%)

E
P

34.3 ± 10.9
30.8 ± 12.8

0.0014 ± 0.0005
0.0015 ± 0.0006

3.13 ± 8.09
3.31 ± 6.77

1.88 ± 1.48*
0.21 ± 0.19

27.6 ± 12.9*
8.3 ± 5.7

41.6 ± 14.1*
11.7 ± 6.9

Values are the mean ± SD (n = 10).
LS, labeling surface; Oc.N, osteoclast number; OS, osteoid surface; OV, osteoid volume; PS, pannus surface; PV, pannus volume; TV, tissue volume.
PV/unit area (TV), PS/TV and Oc.N/PS are not significantly different between the groups. OV/PV, OS/PS and LS/PS increase significantly with etanercept administration.
*P < 0.05 versus the P group.

to be higher in the E group than in the P group (P = 0.053),
and significantly decreased in all groups and at all times
compared with the N group, independent of ETN administration. TNF-alpha, RANKL and OPG mRNA
expressions were not significantly different between E
and P groups at all times. Treatment with ETN tended
to reduce the RANKL/OPG ratio on day 14 (P = 0.072),
but the difference did not reach statistical significanceat any time. DKK-1 mRNA expression in the E groupdecreased significantly on days 7 and 14 (P = 0.009, 0.033,
respectively) compared to the P group, and tended to belower than the P group on day 21 (P = 0.085). With the
exception of day 7 in the E group, expression of Wnt-1
mRNA significantly decreased in all groups and at all
times compared with the N group, independent of ETN
administration. However, ETN administration significantly increased Wnt-1 mRNA expression on days 7 and
21 (P = 0.014, 0.047, respectively) compared with theplacebo administration.

ditions, varied across sections (2–5 fields per section).
Totals of 55 and 53 HPF were examined in the P groupand E group, respectively. The number of plasma cells,
macrophages and blood vessels per HPF were not significantly different between the groups (P = 0.355, 0.355,
0.063, respectively) (Table 3). Histologic sections of theknee joint stained with Villanueva bone stain are shownin Fig. 3. The pannus infiltrated the posteromedial femoral condyle (Fig. 3a). Although pannus formation andbone destruction were not different between the E group(Fig. 3b) and P group (Fig. 3d), more osteoid production(Figs. 3b and d: black arrowheads) and bone calcification (Figs. 3c and e: white arrowheads) were seen on thepannus-contacting bone surface in the E group than in
the P group.
DISCUSSION
Genovese et al. reported that 10% (20 of 201) patientstreated with ETN had negative change in the total
Sharp score from the baseline on 5-year radiographs.22
Klareskog el al. also reported similar results of radiographic changes in RA patients treated with ENT andmethotrexate combination therapy.15 It is clear that development of several effective treatment options such as
ETN has induced repair of bone erosions in RA, but themechanism of “bone repair” is still unknown. To clarify
this uncertain mechanism, we attempted to investigate“bone repair” using molecular biological and histological methods with CIA.
Trentham et al. reported that sensitization to type II
collagen caused arthritis similar to RA.20 CIA has beenused as an RA model for studies on the pathology andtreatment of RA. In the present study, we chose knee
joints because i) the incidence of knee arthritis was high;ii) arthritis of the knee tended to be severe and iii) ourprevious study investigated knee joints of CIA histologically and with molecular biology.21, 23
ETN is a protein that intercepts or inhibits thework of TNF-alpha, but does not inhibit production.3
Therefore, it is acceptable that expression of TNF-alphaExperiment B: Histological findings
Bone histomorphometric analysis of the posteromedialcondyle of the femur in the mid-sagittal plane was performed. The pannus volume and pannus surface were
not significantly different between the groups (P = 0.478,
0.718, respectively) (Table 2). The average number of osteoclasts on the pannus surface did not change with ETN
administration (P = 0.398). Osteoid volume, osteoid surface and labeling surface on the surface of the pannus
increased significantly with ETN administration (P <0.001). The number of HPF, which met the above conTable 3. The number of plasma cells, macrophages andblood vessels per high power field (HPF) in the infiltrating pannus
Group

Plasmacells/HPF

Macrophages/HPF

Blood
vessels/HPF

E
P

0.40 ± 0.33
0.56 ± 0.52

0.26 ± 0.30
0.40 ± 0.43

1.86 ± 0.95
2.33 ± 0.77

Values are the mean ± SD (n = 10).
There are no significant differences between the groups.

17

A. Tanida et al.

Fig. 3. Pannus infiltrated the posteromedial femoral condyle (a). Pannus formation and bone destruction are not different between the Egroup (b, c) and P group (d, e). More osteoid production (b, d: black arrowheads) and bone calcification (c, e: white arrowheads) are seenon the pannus-contacting bone surface in the E group than in the P group (Villaneuva bone stain). a: bar = 500 μm. b, d: bar = 100 μm. c,e: fluorescence, bar = 100 μm. C, cartilage; EL, epiphyseal line; FC, femoral condyle; PC, posterior cortex.

mRNA was not inhibited by ETN administration. Expression of RANKL and OPG were not different withor without ETN administration. However, treatment
with ETN showed a tendency to reduce the RANKL/OPG ratio. Catrina et al. reported that anti-TNF therapy
did not suppress synovial RANKL expression, but promoted OPG expression in RA patients. It was concluded
that therapy with TNF antagonists in RA modulates the
OPG/RANKL system, a potential mechanism that could
explain the retardation of radiographic damage observed
following anti-TNF therapy.24 We think our findings
from an animal model in which anti-TNF therapy reduced the RANKL/OPG ratio in synovial tissue support
their conclusion.
Furthermore, in our histological study, the pannus volume, pannus surface and average number ofosteoclasts on the pannus surface did not change withETN administration. By the same token, the number ofplasma cells, macrophages and synovial blood vessels
were not significantly different among groups. This result is reasonable in the light of our analysis of mRNA
expression that indicated ETN did not suppress RANKL
expression. This implies ETN has limited influence on
osteoclastogenesis.
In the meanwhile, from the standpoint of bone formation, more osteoid and bone calcification were seenon the pannus-contacting bone surface in the E groupthan in the P group. Recently, it was clarified that theWnt family stimulated osteoblast formation via coreceptor LRP5/6, and DKK, an antagonist of LRP5/6,
suppressed osteoblast formation by blocking the Wnt
signaling pathway.10, 25 Interestingly, Diarra et al. reported bone formation in RA was hampered by TNFmediated expression of DKK-1.9 These facts and our results suggest that ETN suppresses DDK-1 expression via
the blocking effect of TNF-alpha and, as a result, Wnt
expression is promoted and osteoblastogenesis becomes
more active independent of regulation of osteoclast activity. Therefore, increase of bone formation is attributed
to the fact that ETN directly promotes osteoblastogenesis and not as a result of suppressing osteoclastogenesis.
Our study has a few limitations. In experiment A,
ETN administration was started 1 day after immunization because TNF-alpha expression began to decrease
after peaking 7 days after immunization.23 A previous
study reported that ETN reduced the incidence of arthritis,26 so one limitation is that ETN administration wasstarted before the onset of arthritis. However, in our preliminary experiment, the incidence of arthritis in CIA
rats was not significantly different between the E group
and P group (85.7%, 89.9% respectively). In experiment
B, ETN administration was started 3 weeks after im18

ETN promotes bone formation
18820702.
12 Bathon JM, Martin RW, Fleischmann RM, Tesser JR, Schiff
MH, Keystone EC, et al. A comparison of etanercept andmethotrexate in patients with early rheumatoid arthritis. N
Engl J Med. 2000;343:1586-93. PMID: 11096165.
13 Genovese MC, Bathon JM, Martin RW, Fleischmann RM,
Tesser JR, Schiff MH, et al. Etanercept versus methotrexate inpatients with early rheumatoid arthritis. Two-year radiographic and clinical outcomes. Arthritis Rheum. 2002;46:1443-50.
PMID: 12115173.
14 Genovese MC, Bathon JM, Fleischmann RM, Moreland
LW, Martin RW, Whitmore JB, et al. Longterm safety, efficacy, and radiographic outcome with etanercept treatment
in patients with early rheumatoid arthritis. J Rheumatol.
2005;32:1232-42. PMID: 15996057.
15 Klareskog L, van der Heijde D, de Jager JP, Gough A, Kalden J,
Michel M, et al. Therapeutic effect of the combination of etanercept and methotrexate compared with each treatment alone
in patients with rheumatoid arthritis: double-blind randomized
controlled trial. Lancet. 2004;363:675-81. PMID: 15001324.
16 Menninger H, Meixner C, Sondgen W. Progression and repair
in radiographs of hands and forefeet in early rheumatoid arthritis. J Rheumatol. 1995;22:1048-54. PMID: 7674229.
17 Rau R, Wassenberg S, Herborn G, Perschel WT, Freitag G.
Identification of radiologic healing phenomena in patients withrheumatoid arthritis. J Rheumatol. 2001;28:2608-15. PMID:
21965641.
18 Sharp JT, van der Heijde D, Boers M, Boonen A, Bruynesteyn
K, Emery P. et al. Repair of erosions in rheumatoid arthritis
does occur. Results from 2 studies by the OMERACT subcommittee on healing of erosions. J Rheumatol. 2003;30:11027. PMID: 12734916.
19 van der Heijde D, Landewe R. Imaging: do erosions heal?
Ann Rheum Dis. 2003;62(Suppl 2):ii10-ii12. doi:10.1136/
ard.62.suppl_2.ii10.
20 Trentham DE, Townes AS, Kang AH. Autoimmunity to type
II collagen: an experimental model of arthritis. J Exp Med.
1977;146:857-68. PMID: 894190.
21 Mizumura H, Nishihara S, Kishimoto Y, Morio Y, Teshima
R. Tartrate-resistant acid phosphatase-positive cells in the
synovial-cartilage junction and bone marrow during the progression of collagen-induced arthritis in adult rats. Mod Rheumatol. 2004;14:123-9. PMID: 17143661.
22 Genovese MC, Bathon JM, Fleischmann RM, Moreland
LW, Martin RW, Whitmore JB, et al. Longterm safety, efficacy, and radiographic outcome with etanerecept treatment
in patients with early rheumatoid arthritis. J Rheumatol.
2005;32:1232-42. PMID: 15996057.
23 Kishimoto Y, Fukumoto S, Nishihara S, Mizumura H, Hirai K,
Teshima R. Gene expression relevant to osteoclastogenesis in
the synovium and bone marrow of mature rats with collageninduced arthritis. Rheumatology. 2004;43:1496-503. PMID:
15353610.
24 Catrina AI, af Klint E, Ernestam S, Catrina SB, Makrygiannakis D, Botusan IR, et al. Anti-tumor necrosis factor therapy
increases synovial osteoprotegerin expression in rheumatoid
arthritis. Arthritis Rheum. 2006;54:76-81. PMID:16385498.
25 Baron R, Rawadi G. Wnt signaling and the regulation of bonemass. Curr Osteoporos Rep. 2007;5:73-80. PMID: 17521509.
26 Shibata H, Yoshioka Y, Abe Y, Ohkawa A, Nomura T, Minowa
K, et al. The treatment of established murine collagen-induced
arthritis with a TNFR1-selective antagonistic mutant TNF.
Biomaterials 2009;30:6638-6647. PMID: 19765818.

munization because bone destruction was observed histologically 3 weeks after immunization.21 As describedabove, if we assume that expression of TNF-alpha may
deteriorate soon after 3 weeks’ post-initial immunization, ETN may be no longer involved in the regulation of
osteoclastogenesis from that point onwards. As a result,bone resorption during histomorfometry was not downregulated. ETN administration was started less than 3
weeks after immunization, which may cause inhibitionof pannus formation itself. This is the second limitationof this study.
Acknowledgments: We acknowledge the Division of Functional
Genomics, Research Center for Bioscience and Technology,
Tottori University for support in gene analysis. We also acknowledge Ms. Akemi Ito for assistance in preparing and staining tissue
sections, and for counsel about histomorphometric analysis.
The authors declare no conflict of interest.
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