Analysis of cartilage-derived

retinoic-acid-sensitive protein (CD-RAP) in

synovial uid from patients with
osteoarthritis and rheumatoid arthritis
S. Saito, S. Kondo, S. Mishima, N. Ishiguro, Y. Hasegawa,
L. J. Sandell, H. Iwata
From Nagoya University School of Medicine, Nagoya, Japan

e have measured the concentration of

cartilage-derived retinoic-acid-sensitive protein
(CD-RAP) in synovial uid (SF) from the knees of 49
patients with osteoarthritis (OA) and 79 with
rheumatoid arthritis (RA) in order to investigate the
correlation between the type of joint disease and level
of CD-RAP. The mean concentration of CD-RAP in
synovial uid was signicantly higher in OA than in
RA. The level of CD-RAP in the group of patients
with mild OA was signicantly higher than in the
moderate or severe groups and that in the group with
mild RA was also signicantly higher than in the
other RA groups and decreased with progression of
the disease. Immunohistochemical studies showed
expression of CD-RAP in the cytoplasm of
chondrocytes in newly-formed brocartilage. Since
CD-RAP is mainly produced in young and
proliferating chondrocytes, our results suggest that the
level of CD-RAP in synovial uid reects remodelling
of articular cartilage and may be used as a marker to
estimate objectively the restorative reaction of
chondrocytes.

J Bone Joint Surg [Br] 2002;84-B:1066-69.

Received 15 February 2001; Accepted after revision 5 April 2002

Cartilage-derived retinoic-acid-sensitive protein (CD-RAP)

is a newly discovered 14 kD secreted protein expressed by
1
chondrocytes. Distribution analysis of CD-RAP mRNA

S. Saito, MD, Orthopaedic Surgeon

S. Kondo, MD, Assistant Professor
S. Mishima, MD, Orthopaedic Surgeon
N. Ishiguro, MD, Associate Professor
Y. Hasegawa, MD, Associate Professor
H. Iwata, MD, Professor and Chairman
Department of Surgery, Nagoya University School of Medicine, 65 Tsurumai-cho, Nagoya 466-8550, Japan.
L. J. Sandell, PhD, Professor
Department of Orthopaedics, Cell Biology and Physiology, Washington
University School of Medicine, St Louis, Missouri 63110, USA.
Correspondence should be sent to Dr S. Kondo.
2002 British Editorial Society of Bone and Joint Surgery
0301-620X/02/712177 $2.00
1066

has shown that during development in the mouse it is

primarily expressed by chondroblasts and chondrocytes,
and appears to be more chondrocyte-specic than the other
molecules generally considered to be markers of the chondrocyte phenotype, such as link protein, aggrecan and type1
II collagen. In the mouse, CD-RAP is strongly expressed
2
in the growth plate but is low in articular cartilage. Its
function in chondrocytes remains unknown except that it
3
inhibits proliferation. In malignant tissues, CD-RAP is
4,5
6
also produced by chondrosarcoma, melanoma and mam7
mary carcinoma. CD-RAP is the same molecule which has
been reported as melanoma inhibitory activity (MIA) and
which was independently isolated from a metastatic melanoma cell line and so named because of its ability to inhibit
8
DNA synthesis in such cell lines. Recently, it was reported
that CD-RAP/MIA is a useful serum marker for the detec9
tion of metastasis of melanoma. Since the expression of
1-3
CD-RAP is restricted in chondrocytes in normal tissue, it
may provide a means of monitoring the metabolic activity
of cartilage.

Patients and Methods

We studied 49 patients with osteoarthritis (OA) of the knee
and 79 with rheumatoid arthritis (RA). Of those with OA,
29 (mean age 61.7 years) had mild, 12 (mean age 67.1
years) moderate and 8 (mean age 69.0 years) severe OA. In
the group with RA, 13 patients (mean age 61.3 years) had
mild, 18 (mean age 63.1 years) moderate and 48 (mean age
65.0 years) severe RA.
All the patients were divided into three groups of mild,
moderate or severe according to classications presented in
10,11
previous reports.
Briey, the evaluation of joint
destruction on plain anteroposterior radiographs and MRI
was performed separately by each of three orthopaedic
surgeons (SS, SK and MS), who had previously been
instructed by the same radiologist. The mean of each
grading repeated three times was compared among the
three surgeons, and when discrepancies were noted, a grade
was assigned after consensus had been reached.
Samples of synovial uid (SF) were aspirated from the
knee of all the patients as part of their therapeutic programme. The SF was centrifuged at 3000 rpm for 20
minutes and the samples frozen and stored at 80C until
THE JOURNAL OF BONE AND JOINT SURGERY

ANALYSIS OF CD-RAP IN SYNOVIAL FLUID FROM PATIENTS WITH OSTEOARTHRITIS AND RHEUMATOID ARTHRITIS

Results
Concentrations of CD-RAP in SF. We compared the
concentration of CD-RAP in samples of SF from patients
with OA and RA. The mean concentration of CD-RAP was
27.7 5.9 ng/ml in the group with OA and 10.87 8.6 ng/
ml in those with RA. The CD-RAP level in OA was
signicantly higher than that in RA (p < 0.01; Fig. 1). The
level of CD-RAP in the group with mild OA was signicantly higher than that in the moderate and the severe
VOL. 84-B, NO. 7, SEPTEMBER 2002

p<0.01

60

CD-RAP concentration (ng/ml)

50
40
30
20
10
0

OA

RA

Fig. 1
Box plots of the percentile distribution of the concentration of CD-RAP in
OA and RA. From the bottom up, the box indicates the 25th, 50th
(median) and 75th percentiles, while the bars indicate the 10th and 90th
percentiles, respectively. Open circles represent individual outliers, less
than 10% in each direction. The level of CD-RAP in OA is signicantly
higher than in RA.

p<0.01
p<0.01
60
CD-RAP concentration (ng/ml)

analysis. Quantitative analysis of CD-RAP in SF samples

was performed using one-step ELISA with anti-human CDRAP antibodies provided by Boehringer-Mannheim
(Mannheim, Germany). Samples (20 l) or CD-RAP standard were incubated with 200 ml of reagent containing the
antibodies in streptavidin-coated microtitre plates for 90
minutes with continuous shaking. After washing three times
in phosphate-buffered saline (PBS), 200 l of 2,2'-aminodi-(3) ethylbenz-thiazoline sulphate (Boehringer-Mannheim) were added to the wells and absorbance was
measured at 405 nm.
Articular cartilage, subchondral bone, bony spur and
synovium were obtained during knee arthroplasty surgery.
The specimens were xed in 10% formalin at room temperature for 24 hours, then embedded in parafn. Deparafnised sections were immersed three times in PBS for
ve minutes each. To show the localisation of CD-RAP,
staining with Alcian Blue was performed. For immunostaining of CD-RAP, the activity of endogenous peroxidase
was blocked by incubation with 0.2% hydrogen peroxide in
methanol for 30 minutes and rinsed in PBS. The slides
were then soaked in normal goat serum for 15 minutes for
blocking and pretreated with 0.04% trypsin in PBS. The
sections were incubated for 18 hours at 4C with biotinylated CD-RAP antibody. After rinsing with PBS, they
were treated by peroxidase-labelled streptavidin (Dako,
Kyoto, Japan) for one hour at room temperature.
The reaction products were developed by immersing the
sections in 3,3'-diaminobenzidine tetrahydrochloride solution containing 0.03% hydrogen peroxide. The nuclei were
counterstained with Mayers haematoxylin. For negative
controls, the primary antibody was substituted with the
antibody diluent. For positive controls, we used chondrosarcoma, which was stained positively (data not
shown).
Statistical analysis. All values were expressed as the mean
standard deviation (SD). The Mann-Whitney U test was
used for comparison between OA and RA groups and the
Kruskal-Wallis test to determine whether concentrations
differed signicantly among the three groups of OA and
RA. If a difference was noted the Sheffe test was used to
compare the groups as a post-hoc test. A p value of < 0.05
was considered to be signicant. Analysis of the data was
performed using StatView 5.0 J software (SAS, Cary, North
Carolina) on a personal computer.

1067

50
40
30
20
10
0

Mild

Moderate

Severe

Fig. 2
The concentration of CD-RAP in SF with OA. The level of CD-RAP in SF
from the group with mild OA was signicantly higher than that in the
other OA groups.

groups (p < 0.005; Fig. 2). In the group with RA, the level
in the mild group was also signicantly higher than that in
the other groups (p < 0.01) and decreased with progression
of the disease (p < 0.05; Fig. 3).
Immunolocalisation of CD-RAP. In order to determine
the origin of CD-RAP in SF, we performed an immunohistochemical study on the specimens of articular cartilage, bony spur and synovium obtained during knee
arthroplasty. Expression of CD-RAP was observed in the
cytoplasm of chondrocytes in the bony spur (Fig. 4), but
very little was detected in the extracellular matrix. There

1068

S. SAITO, S. KONDO, S. MISHIMA, N. ISHIGURO, Y. HASEGAWA, L. J. SANDELL, H. IWATA

p<0.01
p<0.05

p<0.05

CD-RAP concentration (ng/ml)

60
50
40
30
20
Fig. 4a

10
0

Mild

Moderate
Fig. 3

Severe

The concentration of CD-RAP in SF with RA. The level of CD-RAP in SF

from the group with mild RA was highest and decreased as the disease
progressed.

was no expression in either articular cartilage or synovium (data not shown).

Fig. 4b

Discussion
This is the first report to show the existence of CD-RAP in
synovial fluid and the possibility of using the level of CDRAP as a joint marker. In order to assess damage to articular
cartilage, several joint markers have been investigated.
Type-II collagen is mainly expressed in chondrocytes and is
the main component of articular cartilage. C-terminal typeII collagen propeptide is excised from type-II procollagen
during the formation of type-II collagen, released to the
extracellular matrix and finally excreted to the joint fluid.
12
Shinmei et al reported that the level of C-terminal type-II
collagen propeptide in SF in OA is significantly higher than
that in RA, reflecting biosynthesis of collagen in cartilage.
Other studies have indicated that matrix metalloproteinase
(MMP)-3 may be the most important degradative enzyme in
13,14
since it degrades proteoglythe breakdown of cartilage
cans and can activate other MMPs such as collagenases.
MMP-3 in SF is mainly produced by synovial cells and is
15
thought to be a marker for inflammation. Proteoglycan is
also the second component of articular cartilage in the
16
extracellular matrix. Lohmander et al showed that the
level of proteoglycan in SF is very high immediately after
17
injury to the anterior cruciate ligament. Shinmei et al
reported that the level of chondroitin sulphate isomer and
chondroitin sulphate 4S:chondroitin sulphate-6S reflects the
metabolism of proteoglycan in articular cartilage and could
be used to diagnose joint diseases and to predict destruction
of articular cartilage.
We have shown that the level of CD-RAP in SF is more
sensitive and more specific than the joint markers pre-

Fig. 4c
The expression of CD-RAP in newly formed brocartilage. Figure
4a Alcian Blue staining 20. Figure 4b CD-RAP immunostaining 100. Figure 4c CD-RAP immunostaining 400. Fibrocartilage of the bony spur in OA was immunostained with CD-RAP
antibody. Expression was observed in the cytoplasm of chondrocytes
(A, articular surface; B, bone; C, cartilage).

viously reported. Sakano et al showed that CD-RAP is

expressed in proliferating chondrocytes in growth plate
and callus induced by fracture, but not in normal articular
cartilage. However, when cartilage is damaged, the production of CD-RAP is induced to aid the remodelling of
cartilage. Type-II collagen is cartilage-specific and abundantly produced in normal chondrocytes. Its existence in
normal cartilage decreases its sensitivity and specificity
as a joint marker. Since MMPs and proteoglycans are
produced not only by chondrocytes but also by synovial
cells, they were thought to be markers of inflammation
15
rather than of cartilage metabolism. To date, the funcTHE JOURNAL OF BONE AND JOINT SURGERY

ANALYSIS OF CD-RAP IN SYNOVIAL FLUID FROM PATIENTS WITH OSTEOARTHRITIS AND RHEUMATOID ARTHRITIS

tion of CD-RAP remains unknown and there is no

information as to whether it has an effect on inflammation and/or the destruction of cartilage. In our results the
SD is low compared with that in other studies of this type.
We used not only radiography but also MRI to define the
groups. Radiography shows only bone and morphology
but MRI provides information on the quality of the bone
marrow and cartilage. This may explain the low value of
the SD.
CD-RAP is the same molecule as has been reported as
melanoma inhibitory activity (MIA). CD-RAP/MIA is
7,17,18
and
detected in melanoma and mammary carcinoma
9
can be used to monitor activity of the tumour. Increased
serum concentrations were found in rheumatic diseases
19
associated with joint destruction. Recently, Neidhart et
20
al have reported that after marathon running, the serum
level of CD-RAP/MIA is significantly increased. They
suggest that CD-RAP/MIA is a marker for certain aspects
of joint metabolism and damage in sport.
We have shown that the level of CD-RAP in synovial
fluid is sensitive and specific to the degree of joint
disease. We consider that expression of CD-RAP reflects
repair of articular cartilage and decreases with destruction of the cartilage. The concentration of CD-RAP may
be useful for monitoring the degree of restorative
reaction.
This work was supported by a Research grant from the Ministry of Health
and Welfare, and Meiseilcai Foundation.
No benets in any form have been received or will be received from a
commercial party related directly or indirectly to the subject of this
article.

References
1. Dietz UH, Sandell LJ. Cloning of a retinoic acid-sensitive mRNA
expressed in cartilage and during chondrogenesis. J Biol Chem
1996;271:3311-6.
2. Sakano S, Y Zhu, Sandell LJ. Cartilage-derived retinoic acidsensitive protein and type II collagen expression during fracture
healing are potential targets for Sox9 regulation. J Bone Miner Res
1999;14:1891-901.
3. Kondo S, Cha SH, Xie WF, Sandell LJ. Cytokine regulation of
cartilage-derived retinoic acid-sensitive protein (CD-RAP) in primary
articular chondrocytes: suppression by IL-1, bFGF, TGFand stimulation by IGF-1. J Orthop Res 2001;19:712-9.

VOL. 84-B, NO. 7, SEPTEMBER 2002

1069

4. Bosserhoff AK, Kondo S, Moser M, et al. Mouse CD-RAP/MIA

gene: structure, chromosomal localization and expression in cartilage
and chondrosarcoma. Dev Dyn 1997;208:516-25.
5. Chansky HA, Robbins JR, Cha S, et al. Expression of cartilage
extracellular matrix and potential regylatory genes in a new human
chondrosarcoma cell line. J Orthop Res 1997;16:521-30.
6. Blesch A, Bosserhoff AK, Apfel R, et al. Cloning of a novel
malignant melanoma-derived growth-regulatory protein, MIA. Cancer
Res 1994;54:5695-701.
7. Lu J, Pei H, Kaeck M, Thompson HJ. Gene expression changes
associated with chemically induced rat mammary carcinogenesis.
Molecular Carcinogenesis 1997;20:204-15.
8. Bogdahn U, Apfel R, Hahn M, et al. Autocrine tumor cell growthinhibiting activities from human malignant melanoma. Cancer Res
1989;49:5358-63.
9. Bosserhoff AK, Kaufmann M, Kaluza B, et al. Melanoma-inhibiting
activity, a novel serum marker for progression of malignant melanoma. Cancer Res 1997;57:3149-53.
10. Ishiguro N, Ito T, Ito H, et al. Relationship of matrix metalloproteinases and their inhibitors to cartilage proteoglycan and collagen turnover: analyses of synovial uid from patients with
osteoarthritis. Arth Rheum 1999;42:129-36.
11. Ishiguro N, Ito T, Miyazaki K, Iwata H. Matrix metalloproteinases,
tissue inhibitors of metalloproteinases, and glycosaminoglycans in
synovial uid from patients with rheumatoid arthritis. J Rheumatol
1999;26:34-40.
12. Shinmei M, Ito K, Matsuyama S, Yoshihara Y, Matsuzawa K. Joint
uid carboxy-terminal type II procollagen peptide as a marker of
cartilage biosynthesis. Osteoarthr Cartilage 1993;1:121-8.
13. Lohmander LS, Hoerrner LA, Lark MW. Metalloproteinases, tissue
inhibitor, and proteoglycan fragments in knee synovial uid in human
osteoarthritis. Arth Rheum 1993;36:181-9.
14. Clark IM, Powell LK, Ramsey S, Hazleman BL, Cawston TE. The
measurement of collagenase, tissue inhibitor of metalloproteinases
(TIMP), and collagenase-TIMP complex in synovial uids from
patients with osteoarthritis and rheumatoid arthritis. Arthr Rheum
1993;36:372-9.
15. Iwase T, Hasegawa Y, Ishiguro N, et al. Synovial uid cartilage
metabolism marker concentrations in osteonecrosis of the femoral
head compared with osteoarthrosis of the hip. J Rheumatol
1998;25:527-31.
16. Lohmander LS, Dahlberg L, Ryd L, Heinegard D. Increased levels
of proteoglycan fragments in knee joint uid after injury. Arthr Rheum
1989;32:1434-42.
17. Shinmei M, Miyauchi S, Machida A, Miyazaki K. Quantitation of
chondroitin 4-sulfate and chondroitin 6-sulfate in pathologic joint
uid. Arthr Rheum 1992;35:1304-8.
18. Bosserhoff AK, Moser M, Hein R, Landthaler M, Buettner R. In
situ expression patterns of melanoma-inhibiting activity (MIA) in
melanomas and breast cancers. J Pathol 1999;187:446-54.
19. Mller-Ladner U, Bosserhoff AK, Dreher K, et al. MIA (melanoma
inhibitory activity): a potential serum marker for rheumatoid arthritis.
Rheumatology 1999;38:148-54.
20. Neidhart M, Mller-Ladner U, Frey W, et al. Increased serum levels
of non-collagenous matrix proteins (cartilage oligomeric matrix protein and melanoma inhibitory activity) in marathon runners. Osteoarthritis Cartilage 2000;8:222-9.