Path. Res. Pract. 189, 138-143 (1993)

Immunohistochemical Expression of Tenascin in Normal Human Salivary Glands and in Pleomorphic Adenomas

S. Sunardhi-Widyaputra and B. Van Damme Laboratory of Histo- and Cytochemistry, Department of Pathology, Sf. Rafael University Hospital, Faculty of Medicine Catholic University of Leuven, Leuven, Belgium

SUMMARY

The presence of the extacellular matrix glycoprotein tenascin was studied immunohisto-chemically in normal human salivary glands and in pleomorphic adenomas. Its expression was compared to that of fibronectin, and type IV collagen.

In the normal salivary gland, tenascin was found with interruptions in periductal tissues, and continuously in blood vessels, fat cells and around nerve bundles. In pleomorphic adenoma, tenascin was detected surrounding the clusters of epithelioid cells, in areas with a myxoid and a chondroid matrix, and around some myoepithelial cells as a halo. As compared to fibronectin, there is a similar location of tenascin and fibronectin around tumor cell clusters but not in myxoid and chondroid matrices. Fibronectin was found around the cells in chondroid matrix.

In conclusion, tenascin is not only found in malignant tumors but also in benign tumors such as pleomorphic adenoma. The presence of tenascin as a halo around myoepithelial cells suggests a role of these cells in development of myxoid and chondroid matrices.

Introduction

T enascin is a novel extracellular matrix glycoprotein, an oligomer which consists of six disulphide-linked subunits, and which interacts with other extracellular matrix mole-cules and with cells. It has a molecular weight from 190-240 kD in rat and 250-285 kD in human1,3,6-8,13. Tenascin is also known as GP-250, GMEM, Myotendi-neous antigen, Hexabrachion, J 1, gp 150-225, and Cytotactin3,4,6-8, 13, 17, 18,22.

This matrix glycoprotein is found in bone, cartilage and teeth of newborn rats, in the connective tissue directly underlying lingual epithelium, capsular structures sur-rounding the eye and vibrissae of rats and mice32,34. This finding indicates that the biological functions of tenascin are not restricted to hard tissue formation8 Recently tenascin has been suggested to modulate chondrogenic, osteogenic and odontogenic differentiation, to be involved in tumorigenesis, and as it seems to play an important role

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in growth and differentiation, tenascin is also suggested to be involved in epithelial-mesenchymalinteractions8,25,26,3 2. The expression of tenascin in early dental mesenchyme is induced by epithelial tissue33.

In pleomorphic adenoma the epithelial-mesenchymal interactions appear to have an important role in growth and differentiation, especially in producing myxoid and chondroid matrices. It was our aim to study the expression of tenascin in salivary gland pleomorphic adenoma, and to gain an understanding of its role in the tumorigenesis of pleomorphic adenoma.

Material and Methods

Tissue Specimens This investigation is based upon 7 normal human salivary

glands and 20 pleomorphic adenomas which were surgically

1993 by Gustav Fischer Verlag, Stuttgart

removed. All tissues were obtained fresh. A part of each specimen was frozen and stored at - 70C until use. The remainder was fixed, embedded in paraffin and routinely processed. The diag-nosis was made by microscopic examination of routinely stained sections.

Polyclonal Anti-tenascin Antiserum Antiserum to tenascin purified from conditioned medium from

chick and rat embryo fibroblast culture, prepared in rabbits, was kindly provided by Dr. Ruth Chiquet-Ehrismann (Basel, Switzer-land). This antiserum has been shown to cross react with human tenascin 13,14,24.

Immunohistochemical Procedures A Peroxidase Anti-Peroxidase Complex (PAP) method was

used in this work. All procedures were carried out at room temperature. Serial sections of 5 ~m from frozen tissues were mounted on slides and dried overnight, post-fixed in acetone for 10 minutes, and treated with 0.3% H20 2 in methanol for 20 min to reduce endogenous peroxidase activity. The sections were pre-treated with bovine testicular hyaluronidase (Sigma) 0.05 mg per ml PBS for 30 min in order to increase the staining contrast.

The sections were then incubated with antisera against tenascin (diluted 1 : 200) and anti-fibronectin (Dakopatts, diluted 1 : 750) for 30 min, respectively, followed by swine anti-rabbit Ig (Dako-patts, diluted 1: 20), and finally by PAP-rabbit complex (Dako-patts, diluted 1 : 40) for 30 min. The PAP complex was visualized by 3,3'-diaminobenzidine (DAB).

Control sections were incubated without primary antibody, and as positive control we used cryostat sections of human breast carcinoma. The stroma of this tumor has been shown to contain a high amount of tenascin24.

Results

Normal Salivary Gland Figure 1 allows a comparison of the staining patterns

obtained in the normal salivary gland with antibodies to tenascin (Fig. lA), fibronectin (Fig. IB), and type IV col-lagen (Fig. 1 C).

In the normal salivary gland, the expression of tenascin was restricted to stromal tissue around the intercalated ducts, striated ducts, nerve bundles and blood vessels with interruptions of staining around some ducts (Fig. lA).

Tenascin in Pleomorphic Adenomas 139

Some aCllll expressed tenascin staining in parts, while interalveolar and interlobular septae were negative. Fibronectin stained the matrix around the intercalated and striated ducts, blood vessels, around and inside nerve bundles, and in interalveolar septa (Fig. 1 B). Strong positivity for type IV collagen was present around the intercalated and striated ducts, blood vessel walls, around and inside the nerve bundles, and in acinar basement membranes (Fig. 1 C). Both fibronectin and type IV collag-en show a continuous staining at the basal membrane of duct epithelium.

Pleomorphic Adenoma In pleomorphic adenoma tenascin was expressed dif-

fusely in all areas of extracellular matrix of the tumor. Strong positivity for tenascin was found around the solid cell clusters and squamous cell areas (Fig. 2A), myxoid and chondroid matrices (Fig. 3A), and in ductular and trabe-cular tissues (Fig.4A). Within epithelioid cell clusters some cells with dark and irregularly shaped nuclei sur-rounded by an intensely positive halo were observed (Fig. SA and B). These cells were found in each of the specimens examined. This halo formation was not seen for other matrix compounds. We suggest that these cells may be actively growing, modified myoepithelial cells.

Fibronectin in this tumor was seen in parallel to tenascin (Fig.2B, 3B and 4B) but proved to be more strongly positive. Type IV collagen was strongly present around the solid cell clusters, ductal areas, in myxoid and chondroid matrices, in the trabecular and in basement membranes around adenomatous areas (Fig. 2C, 3C and 4C).

Discussion

Tenascin, a novel extracellular matrix glycoprotein, is known to play an important role in tumorigenesis and chondrogenesis, especially in epithelial-mesenchymal interactions8. The presence of tenascin in a growing tissue, both normal and neoplastic, suggests its implication in growth mechanisms. In pleomorphic adenoma, in which chondroid matrix formation is a result of epithelial-mesenchymal interactions, the presence of tenascin may

Fig. 1. Normal salivary gland. Immonoreactivity to tenascin (A) found in the stromal tissues around the intercalated ducts, striated ~ ducts, nerve bundles and blood vessels. In some ducts there were interruptions in tenascin staining. Fibronectin staining (B) and type IV collagen (C) were found more diffusely spread than tenascin, but without interruptions of staining. Cryostat section, PAP with hemalum counterstain (A, B, and C, x 100). - Fig. 2. Pleomorphic adenoma. Squamous cell and epithelioid cell clusters. Immunoreactivity to tenascin (A) expressed diffusely. Strong staining found around this squamous cell and epithelioid areas. Reactivity to fibronectin (B) and type IV collagen (C) seen more strongly positive, in parallel with tenascin staining. Cryostat section, PAP with hemalum counterstain (A and B, x 100, C, X 250).

Fig. 3. Pleomorphic adenoma. Chondroid and myxoid matrices. Immunoreactivity to tenascin (A) is found diffusely and shows a ~ compact staining in the outer part of chondroid areas. Fibronectin (B) and type IV collagen (C) were more strongly positive than tenascin, in the parallel areas. Cryostat section, PAP with hemalum counterstain (A and B, X 100, C, X 250). - Fig. 4. Pleomorphic adenoma. ductular and trabecular areas. Positive staining to tenascin (A) was seen around these parts, in parallel to fibronectin (B), and type IV collagen (C). Cryostat section, PAP with hem alum counterstain (A and B, X 250, C, X 100).

140 . S. Sunardhi-Widyaputra and B. Van Damme

Fig. 1 Fig. 2 ~

Tenascin in Pleomorphic Adenomas 141

Fig. 3 Fig. 4

142 . S. Sunardhi-Widyaputra and B. Van Damme

Fig. 5. Pleomorphic adenoma. Cell with a tenascin halo. In epithelioid cell clusters (A) and in chondroid matrix (B) cells were seen with a tenascin halo (arrows). Cryostat section, PAP with hemalum counterstain (x 400).

provide a clue in finding the cell which produces both chondroid and myxoid matrices.

In our study tenascin is found in different sites in the normal salivary gland and in pleomorphic adenoma. T enascin is a normal component of the peri ductal matrix, similar to the intralobular and periductal matrix of breast tissue, and similar to its presence in the morphogenesis of the salivary gland10,20. In contrast with collagen type IV and fibronectin which stained the complete basal mem-brane, there were some interruptions of tenascin staining. We suggest that this interruption could be related to the discontinuous presence of myoepithelial cells.

The most prominent feature of tenascin reactivity in pleomorphic adenoma was its diffuse presence in all extracellular matrices. This phenomenon may reflect a

continuous production of extracellular matrix. In pleo-morphic adenoma, stromal mucosubstances are composed of glycosaminoglycans, partly hyaluronic acid and partly chondroitin sulphate27,28,31. Tenascin is known to have an ability to interact with chondroitin sulphate proteogly-cans, fibronectin and cells. This complex interaction probably indicates some essential role of tenascin in the organization of the extracellular matrix. Chondroitin sulphate proteoglycans co-precipitate with tenascin (myo-tendineous) antigen35. In mammary carcinogenesis, as well as in pleomorphic adenoma tenascin was detected around dense tumor cells21 . The presence of tenascin in this area suggests tenascin to be involved in epithelial-mesenchymal interactions8.

The presence of cells with a tenascin halo both in epithelioid cell clusters, and in chondroid and myxoid matrices, suggests that the epithelial cells of the pleo-morphic adenoma release tenascin and secrete the glycos-aminoglycans that accumulates intercellularly. In this way epithelial cells become isolated in their ma-trix2, 11, 15, 19,30,31.

Immunoreactivity of fibronectin in pleomorphic adeno-ma occurred in parallel with tenascin, similar to the findings in other tissues I6,25. Tenascin is known to bind to the cell-binding fragment of fibronectin, and both of them are able to influence cell attachment and spreading of mouse fibroblast celllines9.

Other typical stromal components are the type IV collagen around the epithelium, in chondroid matrix and to a limited extent in myxoid matrix, as confirmed previously5, 12, 23, 29.

In conclusion, the presence of tenascin around the dense epithelioid cells suggests a role of the cells with a tenascin halo in producing myxoid and chondroid matrices. The distribution in this tumor merits further study, especially its role in epithelial-mesenchymal interactions and in the formation of myxoid and chondroid matrices.

Acknowledgements

The authors wish to thank Dr. Ruth Chiquet-Ehrismann from Basel, Switzerland, for her generous gift, the anti-tenascin antiserum; the technical assistance of E. Van Dessel and K. Van Meerbeek, and M. Rooseleers for preparation of the micrographs are acknowledged. The author (SS-W) appreciates the financial support from Belgian Administration for Developmental Cooper-ation.

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Received January 27, 1992 . Accepted in revised form April 29, 1992

Key words: Tenascin - Pleomorphic adenoma - Salivary glands - Matrix glycoprotein

S. Sunardhi-Widyaputra, Laboratory of Histo- and Cytochemistry, Department of Pathology, St. Rafael University Hospital, Faculty of Medicine Catholic University of Leuven, Minderbroedersstraat 12, B-3000 Leuven, Belgium