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:minal type II p r o c o l l a g e n peptide as a m a r k e r Lrtilage collagen b i o s y n t h e s i s KAZUHIKOITO$, SI-IIGEOMATSUYAMAt, YASUO YOSHIHARA* AND KIMIHIKO MATSUZAWA$ 'gery and the tCentral Research Laboratory, National Defense Medical Institute for Bio-Medical Research I, Teijin Iwakuni Research Center, Iwakuni, Japan Summary dnal type II procollagen peptide (pCOL II-C) were measured in osteoarthritis, ; arthritis by a newly developed one-step enzyme immunoassay (EIA). The detection ~w as 0.2 ng/ml. The levels of pCOL II-C were significantly (P < 0.001) higher in .tis than in rheumatoid arthritis. In osteoarthritis, pCOL II-C levels were higher in type H collagen is a unique component of cartilage, pCOL II-C levels in joint fluids Tof type H collagen of chondrocytes in the diseased joint and therefore could be II collagen synthesis in articular cartilage in joint diseases. II procollagen peptide, Joint fluid, Collagen biosynthesis, Cartilage.
Introduction
Ruggiero etal. [10] reported t h a t pCOL II-C remains associated with or incorporated into the newly synthesized collagen fibrils. The destruction of articular cartilage is common and is the most crucial problem in arthritides such as osteoarthritis, rheumatoid arthritis and joint trauma. Although the metabolic activity of the chondrocytes of adult articular cartilage is thought to be very low, chondrocytes of osteoarthritic and mechanically injured cartilage can be activated and the metabolic rate of the synthesis and degradation of the matrix is accelerated [11-13]. Indeed, collagen synthesis in moderately degenerated cartilage in h u m a n OA is increased at least five-fold by comparison with normal cartilage [1]. Floman et al. [14] and Eyre et al. [15] obtained similar results from an animal model of osteoarthritis. Their data also demonstrated t h a t most of the collagen synthesized was type II rather than type I. The development of cartilage markers to provide information on collagen metabolism of diseased cartilage would be useful in estimating the pathological condition of joint diseases. Accordingly, we explored the possibility of using measurements of the pCOL II-C level in joint fluid as one of the markers of change in type II collagen metabolism in diseased cartilage. The purpose of this report is to describe an enzyme immunoassay (EIA) method of measuring pCOL II-C in joint fluid and to demonstrate that the level of pCOL II-C in joint
[1] of the dry weight the most abundant cartilage. Although an one genetically II collagen accounts Lilage collagen [2, 3]. t by chondrocytes as ith noncollagenous ~ypropeptide extenremoved by specific type II collagen is pe II procollagen be chemically stable 1 fact, C h o i e t al. [6] erable amounts of it vine long bones. The rocalcin [7], which identical with pCOL localized in newly has been extracted Lture cartilaginous cheal and laryngeal cleus pulposus [9]. i 18 February 1993. Shinmei, Department of se Medical College, 3-2 ~pan. 121
122
S h i n m e i et al.: pCOL II-C a s a c a r t i l a g e c o l l a g e n m a r k e r
fluid can serve as a metabolic marker of type II collagen synthesis in cartilage.
Materials
and methods
PREPARATION OF pCOL II-C
pCOL II-C was extracted and purified, as described by Choi e t a l . [6], from fetal bovine epiphyseal and growth plate cartilage. The 35 kDa protein (pCOL II-C) was identified by SDS-PAGE using 7.5% gel under reducing conditions. Amino acid analyses of purified pCOL II-C were carried out on a Hitachi 835 amino acid analyzer (Hitachi Ltd., Tokyo). Samples for amino acid analyses were hydrolyzed in 6 M HC1 at ll0°C for 24, 48, and 72 h under N 2. The amino acid sequences of the N-terminal region of pCOL II-C were analyzed using a gas phase protein sequencer (Applied Biosystem model 470A, Foster City, CA). The phenylthiohydantoin amino acids were analyzed by HPLC chromatography (Vista 5000, Varian Techtron Pty Ltd., Victoria, Australia). Using globular proteins as markers in the electrophoresis, Coomassie blue staining demonstrated the migration of one protein band with an apparent molecular weight of 35 kDa [6, 9]. Amino acid composition of this protein was similar to that of the C(II)-propeptide of chick embryo reported by Ninomiya et al. [16]. Sequence analysis of the amino acids of the N-terminal portion of pCOL II-C showed the existence of two different sequences; the ratio of the major to the minor sequence was 3:1. The amino acid sequences of the major and minor fractions were identical from the l l t h to the 28th residues. Furthermore, the sequences of these fractions were the same as those of chick embryo [16] except for the 12th residue, which was alanine instead of valine. ANTIBODIES
A rabbit antiserum against pCOL II-C was prepared utilizing a modification of the method of Choi et al. [6]. On day 0, an emulsified mixture consisting of I mg of bovine pCOL II-C in 10 mM phosphate buffered saline, pH 7.2 (PBS) and 1 ml of Freund's complete adjuvant (Difco, Detroit, MI) was injected subcutaneously into multiple sites in a Japanese white rabbit. On day 19, the animal was injected with 0.45mg of the protein and 0.45 ml of Freund's complete adjuvant. The injection was repeated on day 35. Twelve days after the last injection, the rabbit was exsanguinated. The titer of the antiserum against pCOL II-C was
confirmed by conventional E-" pCOL II-C coated microplate Tokyo) and horseradish pere gated goat anti-rabbit IgG Tokyo). The titer of the antis the dilution factor of anti absorbance equal to that of l used as control. The titer ot 1 : 400 000. IgG was purified fr precipitation with 40% ss sulfate and by Protein Richmond, CA) affinity colum Rabbit anti-pCOL II-C anti[ labeled with H R P (Toyobo, 7 according to the following procedures: (1) Maleimide derivative . . . . II-C F(ab') 2 fragment: The prepared from rabbit anti-p( the method of Nisonoff et al. gel filtration HPLC (TSK-g~ Tokyo). The F(ab') 2 fraction with 2.5rag of MBS N-hydroxysuccinimide este, IL) dissolved in DMF followed by the gel filtrat above to obtain the maleimic anti-pCOL II-C antibody F(ab') 2. (2) Introduction of SH groups to HRP~ lated HRP was prepared from the r e a c t i ~ r ~ - ~ and S-acetyl mercaptosuccinic ~ ~ (Aldrich, Milwaukee, WI) by the ~ ~ Ishikawa et al. [18]. (3) Conjugation of maleimi rabbit anti-pCOL II-C antibody with thiolated HRP: The thiols was combined with 8.6mg of Visking tube, and then the mix trated using polyethylene glyc washing off the polyethylene gl trate in the Visking tube was overnight at 25°C. The concentr to gel filtration HPLC and e k pH 7.2 to obtain the HRP-bound
PREPARATION OF RABBIT ANTI-pCOL II'C A ~ IMMOBILIZED ON POLYSTYRENE BALL~:!!
Polystyrene balls (6.35 mm Immunochemical, Okayama, immersed in 20 #g/ml of rabbil antibody solution in 0.1 M citrate at pH 3.0 and held at 4°C for 16 h the antibody solution, the balls times with PBS and allowed BSA-PBS (w/v) solution for 2 h
Osteoarthritis and Cartilage Vol. 1 No. 2
123
.... Table I Classification of the severity of OA, as based on radiological evaluation
Stage
Bone sclerosis osteophyte
Narrowing
Attrition
Subluxation
+ + + +
< 1/2 > 1/2 Contact Wide contact
-< 3 mm 3-10 mm > 10 mm
--- /+ +
II III IV ~ V
bed t h r e e t i m e s w i t h for 18 h. T h e balls
OF p C O L II-C
,~en in j o i n t fluid w a s Lzyme i m m u n o a s s a y )dy i m m o b i l i z e d on fluids w e r e diluted ,~ 2~/o (BSA (w/v) a n d S o m e 0 . 2 m l of t h e [~P-conjugated antir a g m e n t [2 #g/ml at )2], a n d a n a n t i b o d y w e r e i n c u b a t e d at ~l of the i n c u b a t i o n l was washed three n g 3 ml of w a s h i n g c t i v i t y was a s s a y e d 1bat±on of t h e poly5 mM h y d r o g e n peretramethylbenzidine n a m o t o , J a p a n ) at e. A b s o r b a n c e w a s 1 of 1 ml of 0.5 M
p r i m a r y o s t e o a r t h r i t i s (OA) w e r e f u r t h e r subdivided i n t o f o u r a n d five g r o u p s r e s p e c t i v e l y , b a s e d on r a d i o g r a p h i c findings in t h e f e m o r o - t i b i a l j o i n t [21, 22] (Table I). G e n e r a l i z e d OA a n d chond r o c a l c i n o s i s w e r e e x c l u d e d f r o m this study. In r h e u m a t o i d a r t h r i t i s , all p a t i e n t s w e r e classified as h a v i n g definite or c l a s s i c a l R A or as h a v i n g m o r e t h a n f o u r of s e v e n s y m p t o m s u n d e r t h e n e w c r i t e r i a of t h e A R A [19]. J o i n t effusion, w h i c h o c c u r s a f t e r k n e e t r a u m a , w a s d e s i g n a t e d as t r a u m a t i c a r t h r i t i s (TA). Thus, this g r o u p i n c l u d e d p a t i e n t s w h o h a d j o i n t effusion w i t h o u t definite i n j u r y to t h e m e n i s c u s or c r u c i a t e l i g a m e n t , w i t h m e n i s c a l i n j u r y a n d w i t h c r u c i a t e t e a r w i t h or w i t h o u t m e n i s c a l t e a r [23]. T h e d i a g n o s e s of t h e s e p a t i e n t s w e r e c o n f i r m e d by a r t h r o s c o p y . P a t i e n t ages in t h i s g r o u p w e r e b e t w e e n 21 a n d 45 y e a r s (Table II). N o p a t i e n t h a d b e e n t r e a t e d by i n t r a - a r t i c u l a r i n j e c t i o n of steroid, c h o n d r o i t i n p o l y s u l f a t e ( A r t e p a r o n ~) or super-purified h y a l u r o n i c acid (Arz ®) d u r i n g at l e a s t a 3 - m o n t h period p r e c e d i n g this study. All t h e R A p a t i e n t s h a d b e e n t r e a t e d
Table II Characteristics of patients in whom pCOL II-C concentrations were assessed
)COL II-C OF J O I N T
Diagnosis and radiological stage
Age Female/ No. mean±s.D, male
luted w i t h a n e q u a l .ted on gel-filtration 3 SW), e l u t i n g at a PBS. F r a c t i o n s of .yed b y the s t a n d a r d
Osteoarthritis stage II stage III stage IV stage V R h e u m a t o i d arthritis stage I stage II stage III stage IV Traumatic arthritis
103 39 39 21 4 35 6 12 12 5 24
)ove.
PATIENTS
e classified a c c o r d i n g to A m e r i c a n s s ° c i a t i ° n (ARA) disease c r i t e r i a w i t h r h e u m a t o i d a r t h r i t i s (RA) a n d
64 ± 10 60 ± 9 65 ± 9 72 ± 10 77 ± 5 57_ 12 44 ± 16 44 ± 16 56 ± 11 65 ± 9 35 ± 9
83]20 29/10 31/8 19/2 4/0 27]8 5]1 10/2 10/2 2/3 7/17
Only those patients from whom joint fluid samples could be obtained at the first aspiration are included.
124
Shinmei e t al.: pCOL II-C as a cartilage collagen marker
with some of the anti-rheumatic drugs, for example, nonsteroidal anti-inflammatory drugs (NSAID), steroid or disease modifying anti-rheumatic drugs DMARD. In the OA group, NSAID had been administered in commonly used dosages. Aspiration of joint fluid from knee joints was performed under aseptic conditions using No. 21 gauge needles. The effusions were collected in sterile tubes and allowed to stand in an ice bath followed by centrifugation at 10 000 rpm for 10 min to remove all cells and debris. The supernatants were stored at - 2 0 ° C until use. STATISTICAL
14
Synovialf l d ~ (OA)
10;
0
Results One-step sandwich EIAs were performed using rabbit polyclonal anti-bovine pCOL II-C antibodycoated polystyrene balls. The standard curve was linear at concentrations ranging from 0.2 ng/ml to 20 ng/ml of pCOL II-C (Fig. 1). Assays of serially diluted joint fluids also demonstrated linearity at dilutions up to 16-fold (Fig. 2).
1-5
o
1.o
if/
< 0.5
,
1.~'5 ~5 s:0
16
ANALYSIS
Differences between groups were analyzed by the Student's t and Cochran's t tests. Data are expressed as mean + standard deviation (X + S . D . ) ; P values less than 0.05 were considered significant.
0'
Synovial flui~
;0
2'o
pCOL II-C concentration (ng/ml) FIc. 1. Standard curve for one-step EIA of pCOL II-C from fetal bovine epiphyseal cartilage.
8 8~
0 6,
•
i,
2[ ~1/,/ 0 ~
x16x8 x4
Synovial f
(RA)
xl
x2
Dilution FIG. 2. Linearity of the present assay. Thr fluid samples were each serially diluted and To assess intra-run and ducibility, 2 RA and 3 0 A joint 10 times each. The coefficients 4.50, 4.44 and 5.49% in OA anc RA, respectively. Recovery adding 2.5-5 ng/ml of pCOL I] joint fluids. The mean percents 84-112% in OA and 84-108% i Interference against the presen pCOL II-C was examined. TI affected by the addition (0.5-5mg/ml), type I or II (2-20 pg/ml), osteocalcin (2.5-5q tive serum. The effect of drugs the t reat m ent of OA and RA also examined. The drugs te diclofenac (l#g/ml), indome tiaprofenic acid (5.0 pg/ml), pb emorfazone (1.5 ~g/ml), so( (5.0 gg/ml) and dexamethazone concent rat i on that is normall administration (the figure ir theses), none of the drugs signiJ assay.
J
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125
20-
15o
o O
0
0
0
r..) m
© 0
O0 0
o 0
)vial dard twoPLC 'ified ) and
t h e OA and RA mt the immuno~s a main peak in [I-C purified from ' immunoreactive rent sizes were, the OA and RA F pCOL II-C anti~mples from each Fig. 4. pCOL II-C ~[ widely between ~re no significant II-C levels with ICOL II-C concen)A, TA, and RA 0.2-19.1) ng/ml, d and 0.99+-1.65 fly (Fig. 4). The RA groups and ;re,~highly signifilificant difference Whilst more than ' T A patients had led 2 ng/ml, only 2 ng/ml (Fig. 4). n OA, TA and RA lg the concentra:ated fluid) was ld 12.2+-17.8 ng, Lces between the
~P
o 0
OA
o
~RA
TA
FIG. 4. pCOL II-C levels in joint fluids. Carboxy-terminal type II procollagen peptide (pCOL II-C) levels in joint fluids, as measured by enzyme immunoassay. OA, Osteoarthritis; RA, rheumatoid arthritis; TA, traumatic arthritis. Transverse bar indicates mean value. See Tables I and II, and Patients in Methods section for
details. groups were similar to those obtained for concentrations, the values within groups varied more widely. In OA patients, the mean value of pCOL II-C in joint fluid at each stage was 3.85 + 3.23 (stage II), 5.79+_3.70 (stage III), 4.40.+_3.10 (stage IV) and 3.33+_1.36 (stage V). The highest value was obtained at stage III. The value decreased with the progression of the disease, but variability was high within groups (Fig. 5). In RA patients, pCOL II-C levels in joint fluids were much lower and there was no correlation between the level and inflammatory activity (ESR or CRP) or radiological stage of RA (data not shown). Discussion
The measurement of antigens related to procollagen in h u m a n serum has been used to evaluate excess deposition or change of turnover of extracellular fibrous components in various chronic diseases. Proteins such as procollagen type I carboxy-terminal extension peptide [24], aminoterminal type III procollagen peptide [25], 7S or NC1 domain of type IV collagen and type VI collagen [26] have been used. In joint fluid, assays
126
Shinmei
et al.: pCOL
II-C as a cartilage
20
15 o o
(.)
i
10 o 8 ~o
0 o
!oo. R O - O
O --O----
oo 8o
o
o
° o
II
III
o
IV OA stage
V
FIG. 5. Relationship between pCOL II-C levels and OA stage. Relationship between radiological stage of osteoarthritis and pCOL II-C levels in joint fluid. Transverse bar indicates mean value. See Tables I, II and Patients in Methods section for details.
of the amino-terminal type III procollagen peptide have already proven useful as a marker of the local inflammatory activity in rheumatoid arthritis [27]. However, assays of pCOL II-C in joint fluid have not yet been systematically evaluated. Choi et al. [6] performed EIA using rabbit antisera against bovine pCOL II-C and confirmed t h at t hei r antisera did not cross-react with either proteoglycan monomer or with link protein after a 64-fold dilution. Hinek et al. [28] and Hinek and Poole [29] used RIA inhibition assay to measure pCOL II-C production in chondrocyte culture. The one-step EIA we developed here for the carboxy-terminal peptide of type II procollagen uses rabbit polyclonal antibody against the bovine carboxy-terminal segment of type II procollagen, which cross-reacts significantly with the samples containing pCOL II-C from various species [30]. In this method, there is no significant increase in bound peroxidase activity with type I and II collagen, hyaluronate, osteocalcin and IgG-RA factor. The pCOL III-N had no significant effect on our pCOL II-C assay at the level which is expected in RA joint fluid [30]. Drugs used frequently in the t r e a t m e n t of arthritis also had no effect on the
collagen
marker
assay. As the detection limi 0.05 ng/ml in the assay tube, tl joint fluid pCOL II-C is 0.2-2 applicability of pCOL II-C ass also ascertained by accuracy ments. Intra- and inter-ass minimal. The recovery of bov to joint fluid was 84-112%. Since a major source of typ fluid is articular cartilage, pC is believed to be derived fron Higher levels of pCOL II-C wq fluids of OA and TA patients, of RA patients (Fig. 4). As th joint fluid reflects the degr~ pCOL II-C from articula: chondro-osseous spur in wh type II collagen may be stim cate an increase in collagen s cytes. In fact, the existence ( human osteoarthritic ca~ confirmed by Goldwasser et et al. [32]. Furthermore, Cheu in rabbit experiments t hat cartilage defects were repaire sprouting from the subcho~ repaired cartilage synthesiz, Thus, it is conceivable t h a t t of type II collagen is stimuD the increased biomechanice meniscal t ear or by instabi ligament insufficiency. On the other hand, the dep reduced in osteoarthritic cart Lust [35] found an increas procollagen in degenerative c spontaneous, early onset ost~ results suggest t hat newly collagen in osteoarthritic cm rive in the processing of fib easily solubilized into the joi The decrease of pCOL II-C i 4) indicates either a decrease collagen or a fast t u r n o v e r of the latter possibility is not value of standard pCOL II-C ( change before and after incu fluids. Osteoarthritis is a commo: adults and is often asymptorr Several molecules r e l a t e d glycan, such as core protein sulfate [39], can be used to conditions in diseased cartilt are t h o u g h t to reflect primar cartilage. Unlike these techl
Osteoarthritis and Cartilage Vol. 1 No. 2
~
e s c r i b e d in this s t u d y m a y er i m p o r t a n t aspect of cartiii
nowledgments ~
,
~
d
14.
15.
a n k A k i e M a r u t a for h e r techD r M. W. S c h e i n for r e v i s i n g erlp •
16.
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