- Email: [email protected]
An immunohistochemical and in situ hybridization study of the expression of tenascin in synovial membranes from human temporomandibular joints with internal derangement
Archs oral Biol. Vol. 41, No. 11, pp. 1081-1085, 1996
Pergamon PII: S0003-9969(96)00088-X
© 1997ElsevierScienceLtd. All rights reserved Printed in Great Britain 0003-9969/96$15.oo+ o.oo
SHORT COMMUNICATION
AN I M M U N O H I S T O C H E M I C A L AND IN SITU H Y B R I D I Z A T I O N S T U D Y OF T H E E X P R E S S I O N OF T E N A S C I N IN SYNOVIAL M E M B R A N E S F R O M H U M A N T E M P O R O M A N D I B U L A R JOINTS W I T H I N T E R N A L DERANGEMENT HIROAKI YOSHIDA, I'* TOSHIMICHI YOSHIDA, 2 TADAHIKO IIZUKA, ~ TERUYO SAKAKURA 2 and SHIGEYUKI FUJITA ' 'Department of Oral and Maxillofacial Surgery, Faculty of Medicine, Kyoto University. 54, Syogoin, Kawahara-cho, Sakyo-ku, Kyoto-shi 606, Japan, and 2Department of Pathology, School of Medicine, Mie University, 2-174 Edobashi, Tsu, Mie 514, Japan (Accepted 13 August 1996)
Summary--In samples of 27 human temporomandibular joints (internal derangement, ankylosis and control speciraens) the distribution of immunoreactive tenascin was restricted to the walls of blood vessels, at the perineurium, and at the surface of the disc. Tenascin staining was intense in the stroma of hypertrophic synovial membranes, particularly on the surface of severely hypertrophic synovial membranes with i~flammation, proliferation, irregular lining structures and new capillary growth. In situ hybridization of fresh discs and synovial membranes demonstrated that synovial cells as well as fibroblasts and endothelial cells expressed tenascin mRNA. The expression of tenascin mRNA was most evident in synovial cells. It is concluded that synovial cells in the synovial membrane produce tenascin in tile diseased human temporomandibular joint. © 1997 Elsevier Science Ltd. All rights reserved Key words: tenascin, immunohistochemistry, in situ hybridization, temporomandibular joint, synovial membrane.
Tenascin is an ex'Lracellular matrix glycoprotein, composed of six similar subunits joined at their amino terminals by disulphide bonds in a central globular region. The amino acid sequence of tenascin subunits includes epidermal growth factor-like repeats, followed by fibronectin type III homology repeats and a domain with a sequence similar to that of fibrinogen at the carboxy terminal (Cutolo et al., 1992). Tenascin has been localized immunohistochemically in nerves, kidneys, tendons, muscle, ligaments, cartilage and the periosteal layer of developing bone (Crossin et al., 1986). Tenascin is also expressed in the stroma of various epithelial tumours, as well as in healing wounds and granulation tissues (Mackie et al., 1987, 1988; Natali et al., 1990, 1991; Laywell et al., 1992; Moch et al., 1993; Luomanen and Virtanen, 1993; Hauptmann et al., 1995; Ilunga et al., 1995). Tenascin is reportedly produced by mesenchymal and epithelial cells, and *To whom all correspondence should be addressed (Tel: 0081-75-751-3405; Fax: 0081-75-761-9732). Abbreviations: PBS, phosphate buffered saline.
immediately secreted into the extracellular matrix (Erickson and Bourdon, 1989). Arthroscopic surgery and histological analysis of surgical specimens after discectomy have shown synovitis and pathological changes in the human temporomandibular joint (Hall et al., 1984; Castelli et al., 1985; Takaku, 1990). The relation between the degree of synovitis and the clinical symptoms of internal derangement of the joint has not been studied in detail (Murakami et al., 1991; Yoshida et al., 1995). We have now examined immunohistochemically the distribution of extracellular matrix and tenascin in specimens of human temporomandibular joints. We have also sought to identify the cellular source of tenascin in fresh surgical specimens by in situ hybridization with human tenascin mRNA. The specimens of temporomandibular joint were surgically removed from six male and 11 female patients ranging in age from 20 to 72 years (sample nos 11-27). Control joints were obtained from surgical and autopsy samples (nos 1-10) of individuals successfully treated for malignant tumours with symptom-free temporomandibular joints. Table 1 lists the individuals and tissues. All specimens were
1081
1082
H. Yoshida Table 1. Clinical data of patients studied
Sample no.
Sex
Age (years)
Period between onset and surgery Affected (years) side TMJ sound R
1
F
48
R
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
M F F F F F F F F M F F F F F M M F F F M F F M F M
51 61 61 62 62 87 87 91 91 36 20 21 25 29 32 32 32 36 37 37 40 52 57 64 72 72
R L R L R L R L R L R L R L L R L L L R L L R L L R
36 2 3 10 9 2 4 5 5 3 3 1 2 1 5 3 2
Mouth-opening maximum Displacement Perforation of (mm) of TMJ disc TMJ disc L
38 40 40
+
+ + + + + + + +
3 32 48 5O 41 33 40 40 32 42 42 40 39 25 45 37 18
+
+ + + + +
+
+
+ + + + +
+ + +
TMJ, temporomandibular joint. Nos 1-10, samples of the control cases; no. 11, sample of the ankylosis case; nos 12-27, samples of the internal derangements of TMJ cases. cut sagittally, a n d immediately fixed in PBS containing 4 % p a r a f o r m a l d e h y d e . Sections were cut from paraffin-embedded specimens. Serial sections (5 /~m) were placed on slides a n d dried at 37°C overnight. I m m u n o h i s t o c h e m i s t r y was performed with the rat m o n o c l o n a l a n t i - h u m a n tenascin antibody, RCB-1 (Oike et al., 1990). T o inactivate end o g e n o u s peroxidase, all sections were i n c u b a t e d with m e t h a n o l containing 0.3% H202 for 20 rain. T h e sections were then i n c u b a t e d with RCB-1 (diluted 1:2000) in a humidified c h a m b e r at 4°C overnight. They were washed t h o r o u g h l y with PBS, i n c u b a t e d with horseradish peroxidase-conjugated, affinity-purified r a b b i t anti-rat i m m u n o g l o b u l i n ( D A K O ) , diluted 1:200, in a humidified c h a m b e r for 1 h at r o o m temperature, a n d then rinsed t h o r o u g h l y with PBS. Finally, the b o u n d i m m u n o -
complex was visualized by immersing the sections in 0.05% M Tris-HC1 (pH 7.6), 0.01 M NAN3, 0.04% (w/v) 3,3'-diaminobenzidine a n d 0.024% (v/v) H202 for 2 min. Negative controls were provided by replacing the p r i m a r y a n t i b o d y with diluted n o r m a l rat serum. The positive control was paraffin sections of h u m a n a d e n o i d cystic carcinoma; the s t r o m a of this t u m o u r is rich in tenascin. There were two characteristic tenascin-staining profiles, specific a n d diffuse. Tenascin was specifically localized in the walls o f b l o o d vessels, at the perineurium, at the surface o f the joint, at the fibrous covering m e m b r a n e o n the c o n d y l a r head, in the chondrocytes a n d pericellular area in the disc, a n d in the surface o f severely hypertrophic synovial m e m b r a n e s with i n f l a m m a t i o n a n d pro-
Fig. 1. Histology of the synovial membrane in internal derangement of human temporomandibular joint. Haematoxylin and eosin-stained sagittal section; hypertrophic exchange was found in the synovial membrane (arrows). Bar = 50/lm. Fig. 2. Immunohistochemical staining by tenascin antibody. Tenascin was expressed in the stroma of the synovial membrane (arrows). Bar = 50/~m. Fig. 3. In situ hybridization with digoxigenin-labelled oligodeoxynucleotide probes for human tenascin. An intensely positive mRNA signal was seen on the synovial cells hybridized with the antisense probe (arrows). Bar = 50/lm.
Tenascin expression in synovial membranes
i .
.
.
.
.
O
~h
Figures 1-3.
.
.
1083
1084
H. Yoshida
liferation. Tenascin was diffused within the stroma of hypertrophic synovial membranes (Figs 1 and 2), and/or within the stroma of severely deformed discs. Tenascin was localized in the walls of blood vessels, at the perineurium, and at the surface of the disc in almost all sections (sample 1-27), and in the fibrous covering membrane on the condylar head in control autopsy specimens (nos 3, 4). Tenascin was localized in chondrocytes and the pericellular area of a disc only from the patient with ankylosis (no. 11). Tenascin was distributed on the surface of severely hypertrophic synovial membranes, within the stroma of hypertrophic synovial membranes, and within the stroma of severely deformed discs in samples 12-27. The lack of tenascin either in the surface of the severely hypertrophic membranes or within the stroma of hypertrophic synovial membranes was notable. The diffuse staining profile within the stroma of synovial membranes was evident in the hypertrophic synovial membranes that maintained their synovial cell arrangement. The specific staining profiles were found only in the severely hypertrophic synovial membranes. Sometimes these areas included inflammation, proliferation, irregular lining structures and new capillary growth. We examined 27 fresh surgical specimens by in situ hybridization for the expression of tenascin mRNA. Antisense and sense cRNA probes were prepared by in vitro transcription of human tenascin cDNA (kindly provided by Dr L. Zardi, Genoa), using a DIG RNA labelling kit (SP6/T7; Boehringer Mannheim, Mannheim, Germany). Tenascin mRNA expression was detected by in situ hybridization as described by Tsukamoto et al. (1991) with slight modifications (Ishihara et al., 1995). In control autopsy specimens (samples 1-10), tenascin mRNA was not expressed, but we were able to find tenascin mRNA expression in the surgical specimens (11-27). In 13 of 17 samples, large numbers of synovial cells expressed tenascin mRNA in the hypertrophic synovial membrane (Fig. 3). Also, in four of 17 samples, tenascin mRNA was identified in fibroblasts. In two specimens, vascular endothelial cells were positive for the mRNA. These findings confirm that synovial cells produce tenascin. In normal articular cartilage of the human knee, tenascin is distinctly localized in the walls of blood vessels, whereas small amounts are found in the surface zone and in the synovial membrane. In diseased joints, tenascin is distributed more in both the articular cartilage and the synovial membrane (Salter, 1993). Our immunohistochemical studies also showed that tenascin was localized on the walls of the blood vessels, at the perineurium and on the surface of the temporomandibular joint discs. In addition, in situ hybridization demonstrated the expression of
tenascin mRNA in synovial cells, vascular endothelial cells and at the surface of the discs. Tenascin mRNA was not expressed in the control autopsy specimens. This may be due to the interval of up to 24 h between death and removal of the specimen, as the expression of tenascin mRNA may only be demonstrable in tissues fixed immediately after death. Tenascin is reportedly involved in wound healing in most tissues. The increased production of tenascin in the synovial membrane may reflect its important role in local immune responses and tissue repair (Cutolo et al., 1992). In the hypertrophic synovial membranes that maintained a normal arrangement of synovial cells, tenascin was distributed along and below the synovial membrane. However, in inflamed hypertrophic synovial membranes showing proliferation, an irregular lining structure and new capillary growth, tenascin was distributed on the surface of the synovial membrane. These findings suggest a close relation between tenascin and synovitis, which is succeeded by tissue repair. Where tenascin formed a halo around some synovial cells examined immunohistochemically, these cells may have been actively growing and secreting tenascin. In the same samples, tenascin mRNA could be expressed in the synovial cells. In conclusion, intense staining for tenascin was seen in the synovial membranes with hypertrophy, proliferation and inflammation, and tenascin mRNA was expressed in the synovial cells. These findings suggest that there is tenascin production by synovial cells in the diseased human temporomandibular joint.
REFERENCES
Castelli W. A., Nasjleti C. E., Diaz-Perez R. and Caffesse R. G. (1985) Histopathologic findings in temporomandibular joints of aged individuals. J. Prosthet. Dent. 53, 415-419. Crossin K. L., Hoffman S., Grumet M., Thierry J. P. and Edelman G. M. (1986) Site-restricted expression of cytotactin during development of chick embryo. J. Cell Biol. 102, 1917-1930. Cutolo M., Picasso M, Ponassi M., Sun M. Z. and Balza E. (1992) Tenascin and fibronectin distribution in human normal and pathological synovium. J. Rheumatol. 19, 1439-1447. Erickson H. P. and Bourdon M. A. (1989) Tenascin: an extracellular matrix protein prominent in specialized embryonic tissues and tumors. Annu. Rev. Cell Biol. 5, 71-92. Hall M. B., Brown R. W. and Baughman R. A. (1984) Histologic appearance of the bilaminar zone in internal derangement of the temporomandibular joint. Oral Surg. 58, 375-381. Hauptmann S., Zardi L., Siri A., Carnemollia B., Borsi L., Castellucci M., Klosterhalfen B., Hartung P., Weis J., S6cker G., Haubeck H. and Kirkpatrick C. J. (1995) Extracellular matrix proteins in colorectal carcinomas. Lab Invest. 73, 172-182.
Tenascin expression in synovial membranes Ilunga K. and Iriyam~. K. (1995) Expression of tenascin in gastric carcinoma. Br. J. Surg. 82, 948 951. Ishihara A., Yoshida T., Tamaki H. and Sakakura T. (1995) Tenascin expression in cancer cells and stroma of human breast cancer and its prognostic significance. Clin. Cancer Res. 1, 1035 1041. Laywell E. D., D6rries U., Barstch U., Faissner A., Schachner M. and Steindler D. A. (1992) Enhanced expression of the developmentally regulated extracellular matrix molecule tenascin following adult brain injury. Proc. natl. Acad. Sci. USA 89, 2634~2638. Luomanen M. and Virtanen I. (1993) Distribution of tenascin in healing incision, excision and laser wounds. J. oral Pathol. Med. 22, 41 45. Mackie E. J., Chiquet-Ehrismann R., Person C. A., Inajuma Y., Taya K., Kawarada Y. and Sakakura T. (1987) Tenascin is stromal marker for epithelial malignancy in the mammary gland. Proc. natl. Acad. Sci. USA 84, 4621-4625. Mackie E. J., Hairier W. and Liverani D. (1988) Induction of tenascin in healiag wounds. J. Cell Biol. 107, 27572767. Moch H., Torhorst J., Diirmiiller U., Feichter G. E., Sauter G. and Gudat F. (1993) Comparative analysis of the expression of Lenascin and established prognostic factors in human t:~reast cancer. Path: Res. Pract. 189, 510-514. Murakami K., Segami N., Fujimura K. and lizuka T. (1991) Correction between pain and synovitis in patients with internal derangement of the temporomandibular joint. J. ora!. Maxillofac. Surg. 49, 1159 1161. Natali P. G., Nicotra M. R., Bartolazze A., Motolese M., Casccia N., Bigotti A. and Zardi L. (1990) Expression
1085
and production of tenascin in benign and malignant lesions of melanocyte lineage. Int. J. Cancer 46, 586 590. Natali P. G., Nicotra M. R., Bigotti A., Bartrolazze A., Motolese M., Coscia N. and Zardi L. (1991) Comparative analysis of the expression of extracellular matrix protein "tenascin" in normal human fetal, adult and tumor tissue. Int. J. Cancer 47, 811 816. Oike Y., Hiraiwa H., Kawakatsu H., Nishikai M., Okinaka T., Suzuki T., Okada A., Yatani R. and Sakakura T. (1990) Isolation and characterization of human fibroblast tenascin. An extracellular matrix glycoprotein of interest for developmental studies. Int. J. Dev. Biol. 34, 309-317. Salter D. M. (1993) Tenascin is increased in cartilage and synovium from arthritic knees. Br. J. Rheumatol. 32, 780-786. Takaku S. (1990) Chronic discopathy of the temporomandibular joint with special reference to the pathological studies based on 54 disks removed surgically. Asian J. oral Maxillofac. Surg. 2, 55-62. Tsukamoto T., Kusakabe M. and Saga Y. (1991) In situ hybridization with non-radioactive digoxigenin-11-UTPlabeled cDNA probe: localization of developmentally regulated mouse tenascin mRNAs. Int. J. Dev. Biol. 35, 25-32.
Yoshida H., Fujita S., Furuhara M., Nishida M., Murakami K. and Iizuka T. (1995) Immunohistochemical localization of CD44 in the human TMJ discectomised specimens (a preliminary report). Jpn J. oral Diag. oral Med. 7, 313-318.
Pergamon PII: S0003-9969(96)00088-X
© 1997ElsevierScienceLtd. All rights reserved Printed in Great Britain 0003-9969/96$15.oo+ o.oo
SHORT COMMUNICATION
AN I M M U N O H I S T O C H E M I C A L AND IN SITU H Y B R I D I Z A T I O N S T U D Y OF T H E E X P R E S S I O N OF T E N A S C I N IN SYNOVIAL M E M B R A N E S F R O M H U M A N T E M P O R O M A N D I B U L A R JOINTS W I T H I N T E R N A L DERANGEMENT HIROAKI YOSHIDA, I'* TOSHIMICHI YOSHIDA, 2 TADAHIKO IIZUKA, ~ TERUYO SAKAKURA 2 and SHIGEYUKI FUJITA ' 'Department of Oral and Maxillofacial Surgery, Faculty of Medicine, Kyoto University. 54, Syogoin, Kawahara-cho, Sakyo-ku, Kyoto-shi 606, Japan, and 2Department of Pathology, School of Medicine, Mie University, 2-174 Edobashi, Tsu, Mie 514, Japan (Accepted 13 August 1996)
Summary--In samples of 27 human temporomandibular joints (internal derangement, ankylosis and control speciraens) the distribution of immunoreactive tenascin was restricted to the walls of blood vessels, at the perineurium, and at the surface of the disc. Tenascin staining was intense in the stroma of hypertrophic synovial membranes, particularly on the surface of severely hypertrophic synovial membranes with i~flammation, proliferation, irregular lining structures and new capillary growth. In situ hybridization of fresh discs and synovial membranes demonstrated that synovial cells as well as fibroblasts and endothelial cells expressed tenascin mRNA. The expression of tenascin mRNA was most evident in synovial cells. It is concluded that synovial cells in the synovial membrane produce tenascin in tile diseased human temporomandibular joint. © 1997 Elsevier Science Ltd. All rights reserved Key words: tenascin, immunohistochemistry, in situ hybridization, temporomandibular joint, synovial membrane.
Tenascin is an ex'Lracellular matrix glycoprotein, composed of six similar subunits joined at their amino terminals by disulphide bonds in a central globular region. The amino acid sequence of tenascin subunits includes epidermal growth factor-like repeats, followed by fibronectin type III homology repeats and a domain with a sequence similar to that of fibrinogen at the carboxy terminal (Cutolo et al., 1992). Tenascin has been localized immunohistochemically in nerves, kidneys, tendons, muscle, ligaments, cartilage and the periosteal layer of developing bone (Crossin et al., 1986). Tenascin is also expressed in the stroma of various epithelial tumours, as well as in healing wounds and granulation tissues (Mackie et al., 1987, 1988; Natali et al., 1990, 1991; Laywell et al., 1992; Moch et al., 1993; Luomanen and Virtanen, 1993; Hauptmann et al., 1995; Ilunga et al., 1995). Tenascin is reportedly produced by mesenchymal and epithelial cells, and *To whom all correspondence should be addressed (Tel: 0081-75-751-3405; Fax: 0081-75-761-9732). Abbreviations: PBS, phosphate buffered saline.
immediately secreted into the extracellular matrix (Erickson and Bourdon, 1989). Arthroscopic surgery and histological analysis of surgical specimens after discectomy have shown synovitis and pathological changes in the human temporomandibular joint (Hall et al., 1984; Castelli et al., 1985; Takaku, 1990). The relation between the degree of synovitis and the clinical symptoms of internal derangement of the joint has not been studied in detail (Murakami et al., 1991; Yoshida et al., 1995). We have now examined immunohistochemically the distribution of extracellular matrix and tenascin in specimens of human temporomandibular joints. We have also sought to identify the cellular source of tenascin in fresh surgical specimens by in situ hybridization with human tenascin mRNA. The specimens of temporomandibular joint were surgically removed from six male and 11 female patients ranging in age from 20 to 72 years (sample nos 11-27). Control joints were obtained from surgical and autopsy samples (nos 1-10) of individuals successfully treated for malignant tumours with symptom-free temporomandibular joints. Table 1 lists the individuals and tissues. All specimens were
1081
1082
H. Yoshida Table 1. Clinical data of patients studied
Sample no.
Sex
Age (years)
Period between onset and surgery Affected (years) side TMJ sound R
1
F
48
R
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
M F F F F F F F F M F F F F F M M F F F M F F M F M
51 61 61 62 62 87 87 91 91 36 20 21 25 29 32 32 32 36 37 37 40 52 57 64 72 72
R L R L R L R L R L R L R L L R L L L R L L R L L R
36 2 3 10 9 2 4 5 5 3 3 1 2 1 5 3 2
Mouth-opening maximum Displacement Perforation of (mm) of TMJ disc TMJ disc L
38 40 40
+
+ + + + + + + +
3 32 48 5O 41 33 40 40 32 42 42 40 39 25 45 37 18
+
+ + + + +
+
+
+ + + + +
+ + +
TMJ, temporomandibular joint. Nos 1-10, samples of the control cases; no. 11, sample of the ankylosis case; nos 12-27, samples of the internal derangements of TMJ cases. cut sagittally, a n d immediately fixed in PBS containing 4 % p a r a f o r m a l d e h y d e . Sections were cut from paraffin-embedded specimens. Serial sections (5 /~m) were placed on slides a n d dried at 37°C overnight. I m m u n o h i s t o c h e m i s t r y was performed with the rat m o n o c l o n a l a n t i - h u m a n tenascin antibody, RCB-1 (Oike et al., 1990). T o inactivate end o g e n o u s peroxidase, all sections were i n c u b a t e d with m e t h a n o l containing 0.3% H202 for 20 rain. T h e sections were then i n c u b a t e d with RCB-1 (diluted 1:2000) in a humidified c h a m b e r at 4°C overnight. They were washed t h o r o u g h l y with PBS, i n c u b a t e d with horseradish peroxidase-conjugated, affinity-purified r a b b i t anti-rat i m m u n o g l o b u l i n ( D A K O ) , diluted 1:200, in a humidified c h a m b e r for 1 h at r o o m temperature, a n d then rinsed t h o r o u g h l y with PBS. Finally, the b o u n d i m m u n o -
complex was visualized by immersing the sections in 0.05% M Tris-HC1 (pH 7.6), 0.01 M NAN3, 0.04% (w/v) 3,3'-diaminobenzidine a n d 0.024% (v/v) H202 for 2 min. Negative controls were provided by replacing the p r i m a r y a n t i b o d y with diluted n o r m a l rat serum. The positive control was paraffin sections of h u m a n a d e n o i d cystic carcinoma; the s t r o m a of this t u m o u r is rich in tenascin. There were two characteristic tenascin-staining profiles, specific a n d diffuse. Tenascin was specifically localized in the walls o f b l o o d vessels, at the perineurium, at the surface o f the joint, at the fibrous covering m e m b r a n e o n the c o n d y l a r head, in the chondrocytes a n d pericellular area in the disc, a n d in the surface o f severely hypertrophic synovial m e m b r a n e s with i n f l a m m a t i o n a n d pro-
Fig. 1. Histology of the synovial membrane in internal derangement of human temporomandibular joint. Haematoxylin and eosin-stained sagittal section; hypertrophic exchange was found in the synovial membrane (arrows). Bar = 50/lm. Fig. 2. Immunohistochemical staining by tenascin antibody. Tenascin was expressed in the stroma of the synovial membrane (arrows). Bar = 50/~m. Fig. 3. In situ hybridization with digoxigenin-labelled oligodeoxynucleotide probes for human tenascin. An intensely positive mRNA signal was seen on the synovial cells hybridized with the antisense probe (arrows). Bar = 50/lm.
Tenascin expression in synovial membranes
i .
.
.
.
.
O
~h
Figures 1-3.
.
.
1083
1084
H. Yoshida
liferation. Tenascin was diffused within the stroma of hypertrophic synovial membranes (Figs 1 and 2), and/or within the stroma of severely deformed discs. Tenascin was localized in the walls of blood vessels, at the perineurium, and at the surface of the disc in almost all sections (sample 1-27), and in the fibrous covering membrane on the condylar head in control autopsy specimens (nos 3, 4). Tenascin was localized in chondrocytes and the pericellular area of a disc only from the patient with ankylosis (no. 11). Tenascin was distributed on the surface of severely hypertrophic synovial membranes, within the stroma of hypertrophic synovial membranes, and within the stroma of severely deformed discs in samples 12-27. The lack of tenascin either in the surface of the severely hypertrophic membranes or within the stroma of hypertrophic synovial membranes was notable. The diffuse staining profile within the stroma of synovial membranes was evident in the hypertrophic synovial membranes that maintained their synovial cell arrangement. The specific staining profiles were found only in the severely hypertrophic synovial membranes. Sometimes these areas included inflammation, proliferation, irregular lining structures and new capillary growth. We examined 27 fresh surgical specimens by in situ hybridization for the expression of tenascin mRNA. Antisense and sense cRNA probes were prepared by in vitro transcription of human tenascin cDNA (kindly provided by Dr L. Zardi, Genoa), using a DIG RNA labelling kit (SP6/T7; Boehringer Mannheim, Mannheim, Germany). Tenascin mRNA expression was detected by in situ hybridization as described by Tsukamoto et al. (1991) with slight modifications (Ishihara et al., 1995). In control autopsy specimens (samples 1-10), tenascin mRNA was not expressed, but we were able to find tenascin mRNA expression in the surgical specimens (11-27). In 13 of 17 samples, large numbers of synovial cells expressed tenascin mRNA in the hypertrophic synovial membrane (Fig. 3). Also, in four of 17 samples, tenascin mRNA was identified in fibroblasts. In two specimens, vascular endothelial cells were positive for the mRNA. These findings confirm that synovial cells produce tenascin. In normal articular cartilage of the human knee, tenascin is distinctly localized in the walls of blood vessels, whereas small amounts are found in the surface zone and in the synovial membrane. In diseased joints, tenascin is distributed more in both the articular cartilage and the synovial membrane (Salter, 1993). Our immunohistochemical studies also showed that tenascin was localized on the walls of the blood vessels, at the perineurium and on the surface of the temporomandibular joint discs. In addition, in situ hybridization demonstrated the expression of
tenascin mRNA in synovial cells, vascular endothelial cells and at the surface of the discs. Tenascin mRNA was not expressed in the control autopsy specimens. This may be due to the interval of up to 24 h between death and removal of the specimen, as the expression of tenascin mRNA may only be demonstrable in tissues fixed immediately after death. Tenascin is reportedly involved in wound healing in most tissues. The increased production of tenascin in the synovial membrane may reflect its important role in local immune responses and tissue repair (Cutolo et al., 1992). In the hypertrophic synovial membranes that maintained a normal arrangement of synovial cells, tenascin was distributed along and below the synovial membrane. However, in inflamed hypertrophic synovial membranes showing proliferation, an irregular lining structure and new capillary growth, tenascin was distributed on the surface of the synovial membrane. These findings suggest a close relation between tenascin and synovitis, which is succeeded by tissue repair. Where tenascin formed a halo around some synovial cells examined immunohistochemically, these cells may have been actively growing and secreting tenascin. In the same samples, tenascin mRNA could be expressed in the synovial cells. In conclusion, intense staining for tenascin was seen in the synovial membranes with hypertrophy, proliferation and inflammation, and tenascin mRNA was expressed in the synovial cells. These findings suggest that there is tenascin production by synovial cells in the diseased human temporomandibular joint.
REFERENCES
Castelli W. A., Nasjleti C. E., Diaz-Perez R. and Caffesse R. G. (1985) Histopathologic findings in temporomandibular joints of aged individuals. J. Prosthet. Dent. 53, 415-419. Crossin K. L., Hoffman S., Grumet M., Thierry J. P. and Edelman G. M. (1986) Site-restricted expression of cytotactin during development of chick embryo. J. Cell Biol. 102, 1917-1930. Cutolo M., Picasso M, Ponassi M., Sun M. Z. and Balza E. (1992) Tenascin and fibronectin distribution in human normal and pathological synovium. J. Rheumatol. 19, 1439-1447. Erickson H. P. and Bourdon M. A. (1989) Tenascin: an extracellular matrix protein prominent in specialized embryonic tissues and tumors. Annu. Rev. Cell Biol. 5, 71-92. Hall M. B., Brown R. W. and Baughman R. A. (1984) Histologic appearance of the bilaminar zone in internal derangement of the temporomandibular joint. Oral Surg. 58, 375-381. Hauptmann S., Zardi L., Siri A., Carnemollia B., Borsi L., Castellucci M., Klosterhalfen B., Hartung P., Weis J., S6cker G., Haubeck H. and Kirkpatrick C. J. (1995) Extracellular matrix proteins in colorectal carcinomas. Lab Invest. 73, 172-182.
Tenascin expression in synovial membranes Ilunga K. and Iriyam~. K. (1995) Expression of tenascin in gastric carcinoma. Br. J. Surg. 82, 948 951. Ishihara A., Yoshida T., Tamaki H. and Sakakura T. (1995) Tenascin expression in cancer cells and stroma of human breast cancer and its prognostic significance. Clin. Cancer Res. 1, 1035 1041. Laywell E. D., D6rries U., Barstch U., Faissner A., Schachner M. and Steindler D. A. (1992) Enhanced expression of the developmentally regulated extracellular matrix molecule tenascin following adult brain injury. Proc. natl. Acad. Sci. USA 89, 2634~2638. Luomanen M. and Virtanen I. (1993) Distribution of tenascin in healing incision, excision and laser wounds. J. oral Pathol. Med. 22, 41 45. Mackie E. J., Chiquet-Ehrismann R., Person C. A., Inajuma Y., Taya K., Kawarada Y. and Sakakura T. (1987) Tenascin is stromal marker for epithelial malignancy in the mammary gland. Proc. natl. Acad. Sci. USA 84, 4621-4625. Mackie E. J., Hairier W. and Liverani D. (1988) Induction of tenascin in healiag wounds. J. Cell Biol. 107, 27572767. Moch H., Torhorst J., Diirmiiller U., Feichter G. E., Sauter G. and Gudat F. (1993) Comparative analysis of the expression of Lenascin and established prognostic factors in human t:~reast cancer. Path: Res. Pract. 189, 510-514. Murakami K., Segami N., Fujimura K. and lizuka T. (1991) Correction between pain and synovitis in patients with internal derangement of the temporomandibular joint. J. ora!. Maxillofac. Surg. 49, 1159 1161. Natali P. G., Nicotra M. R., Bartolazze A., Motolese M., Casccia N., Bigotti A. and Zardi L. (1990) Expression
1085
and production of tenascin in benign and malignant lesions of melanocyte lineage. Int. J. Cancer 46, 586 590. Natali P. G., Nicotra M. R., Bigotti A., Bartrolazze A., Motolese M., Coscia N. and Zardi L. (1991) Comparative analysis of the expression of extracellular matrix protein "tenascin" in normal human fetal, adult and tumor tissue. Int. J. Cancer 47, 811 816. Oike Y., Hiraiwa H., Kawakatsu H., Nishikai M., Okinaka T., Suzuki T., Okada A., Yatani R. and Sakakura T. (1990) Isolation and characterization of human fibroblast tenascin. An extracellular matrix glycoprotein of interest for developmental studies. Int. J. Dev. Biol. 34, 309-317. Salter D. M. (1993) Tenascin is increased in cartilage and synovium from arthritic knees. Br. J. Rheumatol. 32, 780-786. Takaku S. (1990) Chronic discopathy of the temporomandibular joint with special reference to the pathological studies based on 54 disks removed surgically. Asian J. oral Maxillofac. Surg. 2, 55-62. Tsukamoto T., Kusakabe M. and Saga Y. (1991) In situ hybridization with non-radioactive digoxigenin-11-UTPlabeled cDNA probe: localization of developmentally regulated mouse tenascin mRNAs. Int. J. Dev. Biol. 35, 25-32.
Yoshida H., Fujita S., Furuhara M., Nishida M., Murakami K. and Iizuka T. (1995) Immunohistochemical localization of CD44 in the human TMJ discectomised specimens (a preliminary report). Jpn J. oral Diag. oral Med. 7, 313-318.