8) and osteosarcoma (Saos-2) cells in the serum of patients with osteoarthritis of the temporomandibular joint

8) and osteosarcoma (Saos-2) cells in the serum of patients with osteoarthritis of the temporomandibular joint

Archives of Oral Biology 44 (1999) 403±414 Detection of speci®c antibodies against human cultured chondrosarcoma (HCS-2/8) and osteosarcoma (Saos-2) ...

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Archives of Oral Biology 44 (1999) 403±414

Detection of speci®c antibodies against human cultured chondrosarcoma (HCS-2/8) and osteosarcoma (Saos-2) cells in the serum of patients with osteoarthritis of the temporomandibular joint Takuo Kuboki a,*, Takako Hattori b, Tsunehisa Mizushima a, Manabu Kanyama a, Takuo Fujisawa a, Atsushi Yamashita a, Masaharu Takigawa b a Department of Fixed Prosthodontics, Japan Department of Biochemistry and Molecular Dentistry, Okayama University Dental School, Japan


Accepted 12 January 1999

Abstract To ®nd speci®c humoral antibodies in sera from patients with temporomandibular joint (TMJ) osteoarthritis (OA), an immortal human chondrocyte (HCS-2/8) and osteoblast (Saos-2) cell line derived from a chondrosarcoma and an osteosarcoma, respectively, were used as source proteins of human antigens. Patients with chronically painful TMJ OA (n = 18) but no other joints symptoms were selected from a consecutive series of patients with temperomandibular disorders and sex-matched asymptomatic controls (n = 8) were also recruited. Cellular proteins of the HCS-2/8 and Saos-2 cells were subjected to Western blotting with the OA and control sera as probes. Bandrecognition frequency and the peak optical density of the band were compared between groups by w2 and t-tests. OA sera recognized various bands for the chondrocytes, and one of these (47-kDa) was speci®c for the OA sera. In two OA patients whose treatment outcome was less favorable, the reactivity against the 47-kDa protein was relatively high. In addition, the OA sera clearly cross-reacted with recombinant HSP47. Based on these ®ndings, an autoimmune reaction against chondrocytes could be one of the exaggerating and/or perpetuating mechanisms in the pathophysiology of osteoarthritic TMJs, and the humoral antibody titre against the HSP47-like protein derived from the chondrocytes could be one of the possible markers for the prognosis of the joint pathology. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Autoimmunity; Chondrocytes; HSP47; Serum; Temporomandibular joint

Abbreviations: SDS±PAGE, sodium dodecyl sulphate± polyacrylamide gel electrophoresis, TGF, transforming growth factor. * Corresponding author. Tel.: + 81-86-235-6682; fax: +8186-235-6612. E-mail address: [email protected] (T. Kuboki) 0003-9969/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 0 0 3 - 9 9 6 9 ( 9 9 ) 0 0 0 0 9 - 6


T. Kuboki et al. / Archives of Oral Biology 44 (1999) 403±414

1. Introduction Osteoarthritis of the temporomandibular joint is a degenerative disease that occurs primarily (idiopathically) and/or secondarily to displacement of the articular disc (Westesson and Rolin, 1984; de Bont et al., 1985). According to a longitudinal follow-up study in patients with symptomatic anterior displacement of the disc without reduction (Kurita et al., 1998), approx. 40% of these patients will be free of symptoms in 2.5 years and one-third will experience improvement, whereas one-quarter will continue to be symptomatic because of in¯ammation and degenerative changes in the joint. This ®nding clearly suggests that there is substantial di€erence in the natural course of the disease, presumably due to di€erences in host susceptibility to joint in¯ammation and degenerative changes. From a clinical point of view, it is very important to be able to predict the prognosis of the disease to reduce the possibilities of over- or under-treatment. However, predicting the prognosis by basic clinical examinations and interviews only is dicult. The pathophysiological pathways of in¯ammation in osteoarthritis of the mandibular joint are poorly understood. Prostaglandins and/or prostacyclines are likely to be involved, as non-steroidal anti-in¯ammatory drugs, which inhibit the synthesis of prostaglandin precursors, are symptomatically active in most cases of osteoarthritis. Cellular mechanisms such as cell migration and the release of lysosomal enzymes could also be involved, as shown by the ecacy of local steroids (Kopp et al., 1987). The trigger factor in these synovial in¯ammatory cascades is most probably the release of cartilage degradation products, such as glycosaminoglycans, type II collagen, perhaps via antigenic properties, or crystals of calcium pyrophosphate dihydrate or of hydroxyapatite. Circulating antibodies against components of the extracellular matrix surrounding chondrocytes, such as type II collagen, are apparently present in serum from patients with rheumatoid arthritis (Ste€en, 1970; Andriopoulos et al., 1976; Clague et al., 1981; Trentham et al., 1981; Stuart et al., 1983; Clague and Moore, 1984; Ebringer et al., 1981; Watsson et al., 1986; Fujii et al., 1992). In addition to the extracellular matrix-related autoantibodies, Mollenhauer et al. (1988), Enzman et al. (1990), and Bang et al. (1994) report that serum in rheumatoid arthritis contains speci®c antibodies against cell-surface proteins of chicken sterna or human nasal septal chondrocytes. Sakawa et al. (1996) also reported that these sera speci®cally recognized 105-, 68- and 47-kDa proteins of membrane fractions prepared from an immortal human chondrocyte cell line (HCS-2/8). Hattori et al.

(1998) further isolated the 47-kDa antigen (named RA-A47) speci®cally recognized by serum from the patients with rheumatoid arthritis and reported that it cross-reacted with a monoclonal antibody raised against chick HSP47. In the osteoarthritic hip joint, immune complexes have been described, absorbed on to the cartilage surface and in its most super®cial layer; their presence was correlated with an increased sedimentation rate and rapid progress in joint damage (Cooke et al., 1980). Therefore, it is reasonable to speculate that there might be immunoreactionmediated perpetuation of the osteoarthritic in¯ammation and tissue destruction in chronically painful mandibular joints with severe destruction. But, aside from the accumulating evidence for abnormal autoimmunoreactivity in rheumatoid arthritis, there is little clearly documented evidence of an immune reaction occurring in the chronically painful osteoarthritis mandibular joint. We have now investigated the presence of humoral antibodies to human chondrocyte-derived proteins in the serum of patients with osteoarthritic mandibular joints, and we discuss the relation between the speci®c antibodies and the clinical features.

2. Materials and methods 2.1. Patients and asymptomatic controls Serum samples were obtained from 18 patients who ful®lled the following inclusion criteria: (1) they had consulted the Temporomandibular Disease Clinic of Okayama University Dental School from April 1996 to March 1997 and were diagnosed as having temporomandibular arthralgia; (2) they had complained of temporomandibular joint pain for more than 3 months even with some palliative treatment (intraoral appliances and non-steroidal anti-in¯ammatory drugs); (3) there was tomographic and magnetic-resonance evidence of obvious degenerative changes in the hard tissue of their mandibular joint; (4) they did not complain of pain in other joints. If they were diagnosed as having rheumatoid arthritis according to the American Rheumatism Association criteria established in 1987 (Hert and Spector, 1995), they were excluded from the sample. However, there were three patients who were marginally suspected of having rheumatoid arthritis (Nos. 12, 13, 16) because of eventual elevation of rheumatoid factors and morning sti€ness of the ®ngers. As their symptoms did not involve other synovial joints except for the ®nger sti€ness and had not continued for more than 6 weeks, they did not fully meet the criteria for rheumatoid arthritis. The patients with osteoarthritis were 17 women and one man, mean age

T. Kuboki et al. / Archives of Oral Biology 44 (1999) 403±414

35.5 years (range 13±65 years). Asymptomatic control sera were obtained from eight healthy volunteers, seven women and one man, mean age 23.3 years (range 22±27 years). The control participants were recruited from the students and sta€ of Okayama University Dental School. Inclusion criteria for the asymptomatic controls were: (1) good physical health; (2) no history of pain and dysfunction of the mandibular joints; (3) their maximum ranges of mouth opening (interincisal distance) were more than 40 mm; (4) no joint noise, no condylar translatory restriction and no tenderness found during palpation of the lateral aspect of the mandibular joints in maximum mouth opening and closing cycles. The study protocol was approved by an appropriate committee in our department and informed consent was obtained from each participant before the start of the experiment. 2.2. Serum samples Blood samples were collected by venipuncture and permitted to clot at room temperature for 30±60 min. Serum was separated by centrifugation at 3700 rev/min for 10 min and stored in small portions at ÿ208C until used. 2.3. Cell culture The clonal human chondrosarcoma cell line HCS-2/ 8, which was established by Takigawa et al. (1989) from a well-di€erentiated chondrosarcoma of the proximal portion of the humerus of a 72-year-old Japanese male, was used as an ideal model for normal chondrocytes, because HCS-2/8 cells resemble chondrocytes in that they synthesize cartilage-speci®c matrix components such as types II, IX and XI collagen and aggrecan (Takigawa et al., 1989, 1997; Enomoto and Takigawa, 1992; Tsuji et al., 1996; Tuckwell et al., 1994) and an angiogenesis inhibitor (Ohba et al., 1995), and possess a response pro®le to various vitamins and growth factors similar to that of normal chondrocytes (Enomoto and Takigawa, 1992; Takigawa et al., 1997). They were cultured in Dulbecco's modi®ed Eagle's medium (Nissui Pharmaceutical, Tokyo, Japan) containing 10% fetal bovine serum (Gibco, Grand Island, NY, USA) and incubated at 378C in humidi®ed atmosphere of 5% CO2 in air. The medium was changed twice per week. The human osteosarcoma cell line Saos-2 (Weiss et al., 1986; Rodan et al., 1987) was obtained from the Riken Cell Bank (Tsukuba Science City, Japan) and cultured in a-modi®ed Eagle's medium (Nissui Pharmaceutical) containing 10% fetal bovine serum (Gibco) and incubated at 378C in humidi®ed atmosphere of 5% CO2 in air.


2.4. Western blot Cells were dissolved in 1% SDS and 15 mg of proteins per lane were electrophoresed by 10% SDS± PAGE under reducing conditions. For tests of crossreaction between osteoarthritis sera and HSP47, 0.2 mg of recombinant rat HSP47 (StressGen, Canada) was used per lane. The separated proteins were transferred to a nitrocellulose membrane. After blocking with Tris-bu€ered saline±Tween 20 (TBS-T) (20 mM TrisHCl, pH 7.5, 0.5 M NaCl, 0.05% Tween 20) for 30 min at 378C, the membrane was incubated with TBS-T containing the 1:500 diluted osteoarthritis and control sera for 1.5 hr at 378C, washed with TBS-T solution, and then the membrane was soaked with TBS-T containing 1:3000 diluted alkaline phosphatase-conjugated goat anti-human IgG for 1.5 hr at 378C. The membrane was stained with nitroblue tetrasodium and 5bromo-4-chloro-3-indolyl phosphate. Western blotting for all samples was done at the same time in the same conditions. 2.5. Densitometric analysis The stained membranes were analysed with a onedimensional diversity image-analyszing system (Diversity OneTM; pdi, Inc., NY, USA). After a densitogram had been drawn and background correction made automatically in each lane, number and peak density of the bands recognized were measured as outcome variables. 2.6. Joint imaging In every patient, multiple-layered X-ray tomography and magnetic resonance imaging were used on both mandibular joints. Tomography was done in the sagittal plane at 2-mm thickness and 2-mm intervals to detect hard-tissue deformities, using the Optiplanimat (Siemens Co. Ltd, Germany) with a spiral, 458 pattern. On the multiple-layered tomograms, osteoarthritic changes were classi®ed into three levels (OA1, OA2, and OA3). OA1 represents changes (erosion, sclerosis etc.) with no severe deformity of joint outline; OA2 represents clear outline deformities (¯attening, beaking etc.) without shortening of the condyle; OA3 represents clear condylar shortening with any kind of osteoarthritic change. Joint deformity was estimated by an expert who had no information on the clinical ®ndings and the results from Western blotting. Magnetic resonance scanning was done with the 1.5 T Magnetom (Siemens), with a proton-density and T2-weighted imaging sequence. The scanning plane was set perpendicular to the long axis of the coronal condylar outline and the thickness was 3 mm. The diagnosis of disc position

Fig. 1. Western blotting of the chondrocyte (HCS-2/8) proteins with osteoarthritis (OA) sera. OA sera recognized dozens of proteins, but, a 47-kDa protein (arrow) was more frequently and strongly recognized than in controls. Left-hand side group of lanes (No. 1±18), osteoarthritis; right-hand side (No. 1±8), control. The number for each lane corresponds to the number in Tables 1 and 2.

406 T. Kuboki et al. / Archives of Oral Biology 44 (1999) 403±414

T. Kuboki et al. / Archives of Oral Biology 44 (1999) 403±414


Fig. 2. Frequency of the patients whose sera recognized human chondrocyte-derived proteins in each 10-kDa range. Solid columns, osteoarthritis; open columns, control. Proteins in the range 30±49-kDa were more frequently recognized in the osteoarthritis than the control groups (P < 0.01, P < 0.05, w2 tests).

and subcategorization of the joint pathology were done according to Orsini et al. (1998).

3. Results 3.1. Human chondrocytic cell line HCS2/8

2.7. Clinical examinations and follow-up Clinical examination was done by a structured protocol. Inter- and intra-examiner reliabilities for each item in the protocol were con®rmed as acceptable before the experiment (Matsuka et al., 1997). The clinical items recorded were pain (Visual Analogue Scale of Pain) and range of jaw openings (mm) at the time of serum sampling and follow-up. Pain scores were recorded during rest, maximum opening and chewing. Range of jaw opening was measured at maximum voluntary painless opening (painless), maximum voluntary jaw opening (max), and maximum jaw opening while applying pressure between the maxillary and mandibular teeth with the examiner's ®nger (stretch). After 6 months, the same clinical examination was undertaken to follow any changes in the clinical variables.

Fig. 1 shows the results of the Western blotting with osteoarthritis and control sera as the ®rst antibodies against chondrocyte proteins. The recognition frequency in each 10-kDa segment is shown in Fig. 2. Each control and experimental serum recognized dozens of chondrocyte proteins. From 30- to 39-kDa, and from 40- to 49-kDa, there was a signi®cant di€erence (P < 0.05) in recognition frequency between the osteoarthritic and control participants. In that range a 47-kDa protein was much better recognized in the osteoarthritis sera 83%; (15/18) than in the control (38%; 3/8). The mean peak optical density of this 47kDa band was signi®cantly higher in the osteoarthritic than in the control (P = 0.045). On the other hand, the peaks for the 28-, 32-, and 37-kDa proteins were relatively high in all samples, indicating that they were not speci®c for osteoarthritis. Other characteristic proteins of more than 60-kDa were recognized in each patient, but those individually characterized were also observed in the controls.


T. Kuboki et al. / Archives of Oral Biology 44 (1999) 403±414

Fig. 3. Western blotting of the osteoblast (Saos-2) proteins with osteoarthritis (OA) sera. OA sera recognized dozens of proteins, but, a 47 kDa tended to be more frequently recognized. The number for each lane corresponds to the number in Tables 1 and 2.

3.2. Human osteosarcoma cell line Saos-2 Figs. 3 and 4 show the results of Western blotting and the recognition frequency in each 10-kDa segment of the osteoblast proteins. As described above, from 40- to 49-kDa a relatively large number of the osteoarthritis sera tended to recognize the osteoblast proteins, but there was no statistically signi®cant di€erence in the frequency of recognition between the two groups (w2 test; P = 0.1902). As shown with HCS-2/8, a 47kDa protein from the Saos-2 cells tended to be more frequently recognized by osteoarthritis sera and the

mean peak optical densities of the bands were higher, but the di€erence in optical density between the osteoarthritis and control groups was not signi®cant (onetailed t-test; P = 0.086).

3.3. Cross-reaction of osteoarthritis serum with HSP47 Fig. 5 shows the result of the Western blotting with osteoarthritis serum against rat recombinant HSP47; ®ve sera, which recognized the 47-kDa chondrocyte protein, cross-reacted.

Fig. 4. Frequency of the participants whose sera recognized human osteoblast-derived proteins in each 10-kDa range. Solid columns, osteoarthritis; open columns, control. Proteins in the range 40±49-kDa were frequently recognized in the osteoarthritis than the control groups.

T. Kuboki et al. / Archives of Oral Biology 44 (1999) 403±414


Fig. 5. Western blotting of rat recombinant HSP47 with osteoarthritis (OA) sera. Five OA patients sera that recognized the 47kDa chondrocyte protein cross-reacted to the rat recombinant HSP47. Lane numbers (1±5) represent OA patient sera (No. 4, 5, 6, 7, 8), respectively, and a monoclonal antibody against the recombinant HSP47 is shown as a positive control.

3.4. Relation between chronicity of joint symptoms and peak optical density of the 47-kDa band against HCS-2/ 8 Table 1 shows details of the patients and the diagnosis of joint pathology based on imaging; Table 2 shows signs and symptoms. Among 18 osteoarthritic patients, bilateral degenerative joint changes in tomograms were observed in seven. Non-steroidals were prescribed for 11 patients and an intraoral appliance for eight. Almost all of the degenerative joint pathology was related to anterior displacement of the disc, except for one female patient (No. 5). The peak optical densities of the 47-kDa protein in patients No. 6 and 7 were relatively high for the osteoarthritic group, and these two still had pain at rest in the follow-up period (6 months after serum sampling), even with some aggressive therapies (arthrocentesis twice with hyaluronate injections). Age at sampling (r = 0.4114, P = 0.0898), pain at rest at sampling (r = 0.4377, P = 0.1780), and range of mouth opening (painless) (r = 0.0843, P = 0.7395) had no signi®cant relation to peak optical density in the osteoarthritic patients. There was no correlation between the extent of hard-tissue destruction in the joint and the peak optical density (Table 1).

4. Discussion Measurements of articular-cartilage macromolecules and circulating antibodies, including plasma and joint¯uid concentrations of keratan sulphate (Poole et al.,

1990), hyaluronic acid (Goldberg et al., 1991), and anti-type II collagen antibodies (Stuart et al, 1983), have been used for the local and systemic evaluation of metabolism in articular cartilage and to assess the severity of joint in¯ammation in patients with osteoarthritis and rheumatoid arthritis. Almost all of these studies have focused on the components of the extracellular matrix of chondrocytes, and only a few have been conducted on the antibodies directed against chondrocytes themselves. One reason for this di€erence must be the diculty in obtaining a large number of identical chondrocytes for study, particularly those of human origin. Here, we could fortunately use the cultured human chondrocytes prepared from the cell line HCS-2/8 established by Takigawa et al. (1989). As it is self-evident that to sample of a large amount of articular chondrocytes from patients is very dicult for technical and ethical reasons, we believe our strategy was the most relevant and feasible. Mollenhauer et al. (1988) used Western blotting to determine whether serum samples from patients with rheumatoid arthritis or osteoarthritis of the knee and hip joints recognized chondrocyte surface proteins from chicken sternum. The rheumatoid sera contained 10 antibodies to proteins of between 28- and 155-kDa, while the osteoarthritis sera contained seven antibodies and the control only one, against a 135-kDa protein. They further reported the isolation and characterization of one of the proteins, a 65-kDa (Bang et al., 1994). On the other hand, Enzmann et al. (1990) performed a similar study using cell-surface proteins from human nasal septum as antigens, and found that

Male Female Female Female Female Female Female Female

27 25 22 24 22 22 22 22

Rt Rt Rt Rt Lt Lt Rt

Normal Normal Normal Normal Normal Normal Normal Normal

ADDwoR Normal ADDwoR ADDwoR Normal Normal ADDwoR

+ ÿ ÿ ÿ ÿ ÿ +

ÿ ÿ ÿ + ÿ + + ÿ ÿ + ÿ

Normal Normal Normal Normal Normal Normal Normal Normal

OA2, concavity OA1, er Oa1, ¯, sc OA2, thin, er Normal Normal OA1, ¯

OA3, ¯, er Normal Normal OA2, ¯, er OA3, ¯ OA2, ¯, er OA1, er Normal OA1, er OA2, concavity OA1, ¯

E€usion Tomogram

Normal Normal Normal Normal Normal Normal Normal Normal

Normal Normal Normal ADDwR ADDwoR ADDwoR Normal

ADDWoR ADDwoR ADDwoR ADDwoR Perforation? ADDwoR Normal ADDwoR ADDwoR ADDwoR Normal


Imaging data (lt)

ÿ ÿ ÿ ÿ + + ÿ

ÿ + + ÿ ÿ ÿ ÿ + + + ÿ

Other information

Normal Normal Normal Normal Normal Normal Normal Normal

Grandmother: TMJ pain

Mother: TMJ pain; grandmother: RA Mother: RA

Elder sister: TMJ pain Mother: TMJ noise

Grandmother, RA

Familial aggregation history

NP Atopic dermatitis Atopic dermatitis Allergic rhinitis NP Comedo Father: RP Atopic dermatitis Migraine, infectious mononucleosis

Open bite Migraine Asthema NP Open bite, allergic rhinitis Severe chronic pain of Rt TMJ Chronic marginal periodontitis Migraine Myofascial pain of left shoulder NP Myofascila pain of right back Normal RA suspected, ®bromyalgia Normal RA suspected, allergic rhinitis Normal Atopic dermatitis OA2, thin, lip Myofascial pain of right shoulder OA3, ¯ RA suspected OA1, ¯ NP Normal

OA3, ¯, er OA3, ¯, er OA3,¯, er OA2, ¯, er OS3, ¯ OA2, ¯, er Normal OA1, er OA1, er OA1, thin Normal

E€usion Tomogram

ADD, anterior disc displacement; wR, with reduction; woR, without reduction; RP, rapidly progressive periodontitis; RA, rheumatoid arthritis; Side (CC), chief complaint side; E€usion was diagnosed by T2 weighed MRI; NP, nothing particular; ¯, ¯attening; er, erosion; thin, thinning; sc, screlosis; lip, lipping; OA1, no severe joint-outline deformity; OA2, joints with clear outline deformity but shortening; OA3, joints with clear shortening of the condyle; TMJ, temporomandibular joint.



1 2 3 4 5 6 7 8

Female Female Female Female Female Female Female

65 42 27 48 32 19 42


ADDwoR Normal Normal ADDwor Perforation? ADDwoR ADDwoR Normal ADDwoR ADDwoR ADDwoR

12 13 14 15 16 17 18

Both Lt Lt Rt Both Rt Rt Lt Lt Both Rt

37 19 15 13 46 54 33 31 34 32 50


1 2 3 4 5 6 7 8 9 10 11

Female Female Female Female Female Male Female Female Female Female Female

Age Side MRI (CC)

No. Name Sex

Imaging data (rt)

Table 1 Demographic and diagnostic information of the osteoarthritis (OA) patients and symptomatic volunteersa

410 T. Kuboki et al. / Archives of Oral Biology 44 (1999) 403±414



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

1 2 3 4 5 6 7 8

Male Female Female Female Female Female Female Female

Female Female Female Female Female Male Female Female Female Female Female Female Female Female Female Female Female Female


27 25 22 24 22 22 22 22

37 19 15 13 46 54 33 31 34 32 50 65 42 27 48 32 19 42

Age (years)

0,0,0 0,0,0 0,0,0 0,0,0 0,0,0 0,0,0 0,0,0 0,0,0

0,7,0 ± 0,22,7 0,11,6 0,15,0 60,88,70 2,50,44 0,91,70 66,-,10 0,100,52 0,5,0 60,90,70 48,58,52 Ð 0,72,77 0,46,9 0,14,60 0,84,82

VAS (mm)

58 41 45 42 41 47 45 44

38 41 22 21 44 37 17 25 54 29 35 22 40 40 34 33 21 22


58 41 46 42 42 47 45 44

47 44 24 24 44 44 22 28 54 33 37 27 44 45 38 33 23 24


60 42 48 45 48 48 47 45

47 48 29 26.5 46 45 27 29 54 34 38 29 45 46 39 36 25 26


No No No No No No No No

No PM (AC + HA inj)2 PM No (AC + HA inj)2 (AC + HA inj)2 AC + HA inj No No No No No No No No No PM

Intra-articular therapy

No No No No No No No No


Appliance, medication


0,14,8 0$,26,17 0,17,0 0,40,40 0,10,0 62,94,96 5,15,25 0,6,0 0,5,0 Ð 0,0,0 0,0,34 Ð 0,5,0 0,72,75 0,8,0 Ð 0,29,0

VAS (mm)


44 37 30 31 44 41 27 40 53 Ð 34 37 Ð 45 40 36 Ð 35


Pain level and RJO (mm) at follow-up


44 40 34 36 44 44 35 43 54 Ð 34 37 Ð 47 42 39 Ð 41



54 42 37 37 45 45 42 44 55 Ð 35 39 Ð 50 42.5 40 Ð 43


0.041 0.002 0.121 0.037 0.053 0.024 0.034 0.148

0.076 0.115 0.057 0.051 0.130 0.485 0.204 0.131 0.126 0.120 0.047 0.159 0.058 0.040 0.067 0.046 0.036 0.049

(vs chrondrocyte)

Peak OD

VAS, Visual Analogue Scale of Pain (rest, maximum open, chewing); $, no pain but displeased fealing; RJO, range of jaw opening; OD, optical density; PM, pumping manipulation; AC + HA inj, arthrocentesis and hyaluronate injection; SA, stabilization intraoral appliance; Amitriptyline, tricyctic antidepressant; NSAID, non-steroidal anti-in¯ammatory drug; Carb., carbamazepine; , >mean + 2SD; Ð, no data available.2.




Pain level and RJO (mm) at sampling

Table 2 Signs and symptoms of the osteoarthritic patients and asymptomatic volunteersa

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T. Kuboki et al. / Archives of Oral Biology 44 (1999) 403±414

serum antibody to a 66-kDa protein was present in six out of 10 patients with rheumatoid arthritis and antibody to a 52-kDa protein in four out of 10. In the present study, antibodies against a 47-kDa protein were evident in the sera of patients with osteoarthritis of the mandibular joints. These ®ndings di€er from those of previous studies. Possible reasons for this discrepancy include a di€erence in the site from which human chondrocytes were obtained, or in the origin of the chondrocytes, whether from normal cartilage or chondrosarcoma. The principal reason, however, appears to have been di€erence in species (whether human or not). When the study of antigen±antibody reactions is undertaken with human samples, the species speci®city of the reaction must be taken into consideration, and in fact the ®ndings of studies using di€erent species as source of antigens appear to be incorrect. In the present study, dozens of proteins were recognized by both osteoarthritis and control sera. This may have occurred in part because the blocking methods used in the Western blot were insucient, thus eliciting nonspeci®c reactions. The principal reason for the recognition of some 12 proteins, however, was that the antigens used were of human origin; the number of proteins recognized by both the experimental and control sera was correspondingly higher than that in previous reports. Hattori et al. (1998) isolated two types of 47-kDa antigen speci®cally recognized by sera from patients with rheumatoid arthritis from the membrane fraction of the HCS-2/8 cells. An N-terminal amino-acid sequence in one of the 47-kDa antigens, named RAA47, had 81% homology to that deduced from the DNA sequence of the colligin gene (Clarke and Sanwal, 1992), which is reported as the human hsp47 gene, and 100% homology to that deduced from the DNA sequence of the colligin-2 gene (Ikegawa et al., 1995), a homologue of colligin. The RA-A47 crossreacted with a monoclonal antibody raised against chick HSP47 and bound to gelatin. The expression of the ra-a47 gene was further enhanced by heat-shock treatment and stimulation with TGF-b. It is also known that HSP47 is a collagen-speci®c molecular chaperone: it binds collagen and is induced by heat shock and TGF-b (Nagata, 1996). Based on the ®ndings, Hattori et al. (1998) concluded that RA-A47 is a HSP47-like protein, presumably the product of the colligin-2 gene, and that these collagen-speci®c molecular chaperones might be involved in cartilage destruction in rheumatoid arthritis. In our study the osteoarthritis sera also cross-reacted to HSP47, which strongly suggests that the 47-kDa antigen detected by those sera is the protein RA-A47 or HSP47 reported to be related to the pathology of rheumatoid arthritis by Hattori et al. (1998). However, this assumption needs

further direct validation with a blocking test using recombinant RA-A47 or HSP47. As the collagen-speci®c molecular chaperone is thought not to exist outside of the cells in normal conditions, a `hidden antigen' theory is best ®tted to the pathogenesis of the prolonged articular in¯ammation. By adverse mechanical stresses, the chondrocytes could be damaged and their intracellular contents released to immune-presentation cells. Once the immune reaction against the hidden antigen is established in a particular group of individuals, intra-articular in¯ammation could be prolonged by immune complex-related exaggeration of the in¯ammation and degenerative change. We did not take radiographs of the other joints in all the patients or controls, so we cannot exclude completely the existence of asymptomatic polyarthritis. As shown in Table 1, many osteoarthritic and control participants had immunologically active in¯ammatory conditions, such as atopic dermatitis and chronic marginal periodontitis. As these in¯ammatory conditions were equally prevalent in both groups, it seems unrealistic that the systemic in¯ammation could a€ect the outcome of this study as a confounding factor. However, the peak optical density (vs chondrocyte) of a control (No. 8) was relatively high (0.148) even though she had no pain in the mandibular joints. After a detailed medical interview, we found that she had experienced Epstein±Barr virus infection (e.g. infectious mononucleosis), which is well known to enhance humoral immune reactions for a long time. To collect more direct evidence of the immune reaction-mediated perpetuation of the joint symptoms locally, detecting an antibody against the 47-kDa protein in the synovial ¯uids of the mandibular joint might be a good path for the future research. Our patients and the asymptomatic controls were not fully matched in age; the mean age of the osteoarthritic group was higher than that of the control. There was a possibility that ageing might be a confounding factor in elevating the speci®c serum antibodies in the experimental group. To assess this possibility, the relation between the participant's age and the peak optical density of the 47-kDa band on Western blotting was tested statistically: there was no clear relation between them. This result indirectly suggests that it is dicult to explain the amount of serum antibody by their age only. On the other hand, it is interesting that two patients with osteoarthritis whose sera provoked extremely high peak optical densities had poor prognoses, e.g. continuing pain at rest on the follow-up. It is of great importance that clinicians treating patients with temporomandibular disorders be able to predict the natural course and the probable ®nal extent of joint destruction, and this quantitative analysis related to the autoimmune reaction could become a complementary laboratory test to

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