Tenascin expression in mucocutaneous diseases and related lesions of human oral mucosa

Pergamon 0003-9969(95)00071-2

Archs oral Biol. Vol. 40, No. 11, pp. 1039-1045, 1995 Copyright © 1995 Elsevier Science Ltd

Printed in Great Britain. All rights reserved 0003-9969/95 $9.50 + 0.00

T E N A S C I N EXPRESSION IN M U C O C U T A N E O U S ~

DISEASES A N D R E L A T E D LESIONS OF H U M A N O R A L

M U C O S A

OUTI TIITTA, ~* MARITA LUOMANEN, l JARKKO HIETANEN 2 and ISMO VIRTANEN 1

t Institute of Biomedicine, Department of Anatomy, University of Helsinki, P.O. Box 9 (Siltavuorenpenger 20 A), and 2Department of Oral Pathology, Institute of Dentistry, P.O. Box 41 (Mannerheimintie 172),

FIN-00014 University of Helsinki, Finland

(Accepted 9 M a y 1995)

Summary--The expression of tenascin was assessed immunohistochemically. In normal oral mucosa, immunoreactivity for tenascin was seen either as a delicate line underlining the epithelium or in the stromal papillae. In oral lichen planus, a marked enhancement of tenascin immunoreactivity in the lamina propria was associated with focal infiltrates of inflammatory cells and seemed to reflect the intensity of inflammation. In lichenoid reactions in which only a sparse inflammatory infiltrate was present a band-like tenascin reactivity was seen. Oral psoriform reactions and chronic hyperplastic candidosis showed a prominent tenascin reaction in the connective tissue papillae among infiltrates of inflammatory cells. The results show that tenascin content is increased in oral mucocutaneous diseases and related lesions and that the abundance of tenascin reflects the intensity of the inflammatory reaction.

Key words: ,zxtracellular matrix, tenascin, oral lichen planus, oral candidosis, oral psoriasis.

i[NTRODUCTION

In adult tissues, tenascin is restricted to particular sites, for example, smooth muscle and variably at epitheliat-mesenchymal interfaces (Erickson and Bourdon, 1989; Koukoulis et al., 1991). During embryogenesis it is abundantly expressed at sites of epithelial-mesenchymal interaction (Aufderheide, Chiquet-Ehrismann and Ekblom, 1987; Erickson and Bourdon, 1989). A distinct upregulation of tenascin is seen in inflammation and tissue repair (Mackie, Halfter and Liverani, 1988; Koukoulis et aL, 1991; Luomanen and Virtanen, 1993; Whitby et al., 1991), suggesting that it may function in organizing and remodelling the stroma in normal and pathological processes. Also, in the stromal compartment of various carcinomas, there is distinct expression of tenascin (Chiquet-Ehrismann et al., 1986; Erickson and Bourdon, 1989; Howeedy et al., 1990; Koukoulis et al., 1991; Natali et al., 1991).

Tenascin shares structural homology with epidermal growth factor, fibronectin and fibrinogen (Erickson, 1994; Jones et al., 1988; Siri et al., 1991). There are several isoforms of tenascin, which are alternatively spliced from precursor mRNA (Siri et al., 1991). They are differentially expressed during embryogenesis and tumorigenesis (Prieto et al., 1990; Borsi et al., 1992). Based on early studies, tenascin was considered a product of mainly mesenchymal and glial cells (Chiquet-Ehrismann, 1990). More

*To whom all correspondence should be addressed.

recent studies have shown, however, that epithelial cells are also able to synthesize tenascin (Koch et al., 1991; Linnala et al., 1993; Lightner, Marks and McCachren, 1994; Soini, Lehto and Virtanen, 1993).

Tenascin has been demonstrated in both normal and injured oral mucosa of rat (Luomanen and Virtanen, 1993), mouse (Sloan et al., 1990) and man (Reichart et al., 1994; Tiitta et al., 1994) and in normal and diseased human skin (Schalkwijk et al., 1991a; Shikata et l., 1994). In normal human skin and oral mucosa, its expression is variably limited to the epithelial-stromal interface. In oral fibrous hyper- plasias (Becker, Schuppan and Muller, 1993) and in oral submucous fibrosis (Reichert et al., 1994), tenascin has a similar distribution. In hyperproliferative skin diseases, on the other hand, its expression is enhanced in the dermis (Schalkwijk et al., 1991b).

In a previous study, we found that tenascin expression in normal human oral mucosa is restricted to the basement membrane region, while in preneo- plastic and neoplastic lesions stromal tenascin immunoreactivity is markedly enhanced and is frequently associated with inflammatory cell infiltra- tion (Tiitta et al., 1994; see also Shrestha et al., 1994). Little is known about the expression of tenascin in oral mucocutaneous lesions. We have now investi- gated the oral manifestations of inflammatory mucocutaneous diseases and related lesions of oral mucosa in order to clarify further the association of tenascin with inflammation.

1039

1040 Outi Tiitta et al.

MATERIALS AND METHODS

Tissue samples

Oral biopsy specimens with a diagnosis of lichen planus (n = 13), lichenoid reaction (n = 5), psoriform reaction (n = 2) and candidosis (n = 2) were included in the study, together with other samples of normal oral tissue (n = 4). All the samples were retrieved from the files of the Department of Oral Pathology, Institute of Dentistry, University of Helsinki, Fin- land. Adjacent sections were used for immunohistochemical analysis and for conventional light microscopy with haematoxylin and eosin.

Immunohistochemistry

Paraffin sections were cut at 6/~m, deparaffinized and treated with pepsin (0.04% pepsin in 0.01 M HC1 at 37°C for 30 min). They were then incubated in a humified chamber with the monoclonal antibody 143BD7 against tenascin (Tiitta et al., 1992), followed by alkaline phosphatase-conjugated rabbit immunoglobulins to mouse immunoglobulins (Dako, Glostrup, Denmark).

Monoclonal antibody 143BD7 recognizes both the major isoforms of tenascin as shown by Western blotting (Tiitta et al., 1992). Its specificity against the fibrinogen-like head region of tenascin was kindly tested, using bacterial fusion proteins in an enzyme immunoassay as described (Aukhil et al., 1993), by Dr H. P. Erickson (Department of Cell Biology, Duke University Medical Center, Durham, NC, U.S.A.).

The sections were then incubated with naphthol AS-BI phosphate (Sigma, St Louis, MO, U.S.A.) for colour reaction. Afterwards, the sections were briefly counterstained with Mayer's haematoxylin. For control immunostaining experiments, the primary monoclonal antibody was omitted or replaced with an irrelevant monoclonal against placental protein 5. For immunofluorescence microscopy, the sections were first exposed to the monoclonal 143DB7 and then to fluorescein isothiocyanate-coupled rabbit anti-mouse antiserum (Jackson Laboratories, West Grove, PA, U.S.A.).

cells. In the lamina propria, it was seen around the capillaries and along the perineural sheets.

Oral lichen planus

In biopsies of oral lichen planus, a clearly enhanced tenascin immunoreaction was seen in the lamina propria. It appeared as a well-defined, band-like region in association with inflammatory cell infiltrates (Figs 2 and 3). No immunoreactivity was seen in the connective tissue adjacent to inflammatory foci. There was a distinct demarcation between the normal and affected mucosa (see Fig. 2) and an enhancement of the immunoreaction was seen only in areas of histologically defined lichen planus. Tenascin was especially prominent in regions of liquefaction degeneration (Fig. 3). The immunoreactivity for tenascin was also markedly increased in the connective tissue underlying epithelial ulcerations (Fig. 4). Tenascin-immunoreactive rims were also seen around the capillaries and along nerves.

Lichenoid reaction

In biopsies showing a lichenoid reaction (Fig. 5), a clearly enhanced, band-like tenascin-immunoreactivity was seen in the subepithelial lamina propria. The staining for tenascin was even throughout the lamina propria without any denser foci. A strong immunoreaction was also seen in the ulcerative areas of the lesions (Fig. 6). Vessels in the connective tissue also showed the immunoreaction.

Psoriform reaction

In oral biopsies showing a psoriform reaction (Fig. 7), tenascin immunoreactions were most prominent in the connective tissue papillae between the epithelial rete ridges. The strongly immunoreactive stroma of these papillae contained many small vessels and mononuclear inflammatory cells. The epithelium above the papillae was mostly thin and also showed many inflammatory cells. The lamina propria underneath the long rete ridges showed only a diffuse increase in tenascin immunoreactivity.

RESULTS

Normal mucosa

In normal oral buccal mucosa, tenascin immunoreactivity (Fig. 1) was seen either as a delicate or a patchy line immediately adjacent to basal epithelial

Chronic hyperplastic candidosis

In oral biopsies showing chronic hyperplastic candidosis, a marked tenascin-immunoreaction for tenascin was seen in the connective tissue underlying the acanthotic epithelium (Fig. 8). The connective tissue, with accumulations of inflammatory cells,

(Figs 1-4 Opposite) Fig. 1. In normal oral mucosa, tenascin immunoreaction is seen as a thin, faint line in the connective tissue papillae underlying the epithelium. The insert shows immunofluorescence staining with a delicate

tenascin-immunoreactive line underlying normal oral mucosa. (lp = lamina propria.) × 100.

Fig. 2. Biopsy specimen taken from a patient with lichen planus showing parakeratotic epithelial surface, basal epithelial cell degeneration and band-like lymphocytic infiltrate in the upper lamina propria. A marked tenasc{n immunoreaetion is seen in association with both a typical round-cell infiltrate and

elsewhere. Note the increased tenasein expression especially beneath the affected mucosa, x 100.

Fig. 3. A strong tenascin immunoreactivity is seen in areas showing liquefaction degeneration, x 150.

Fig. 4. Lichen planus with epithelial ulceration--massive tenascin immunoreactivity in the lamina propria. x 100.

Tenascin in mucocutaneous lesions 1041

Figs 1 4 . Captions opposite.


showed a strong immunoreaction. The most intensive staining was seen in the stromal papillae underlying those areas that showed intraepithelial inflammatory cells.

Control immunostaining experiments

In the control experiments, the immunoreactions were compared with consecutive sections reacted with an irrelevant antibody followed by the alkaline phosphatase conjugate. As demonstrated in Fig. 9 with specimens from a psoriform reaction (a, b) and lichen planus (c, d), the alkaline phosphatase reaction can easily be distinguished from the background nuclear staining of even dense aggregates of lymphoid cells.

DISCUSSION

In normal human oral mucosa there is only an occasional, sparse expression of tenascin in the basement membrane region (Becker et al., 1993; Reichert et al., 1994; Tiitta et al., 1994). We found clearly enhanced expression of tenascin in the lamina propria of oral mucosa in association with oral mucocutaneous diseases and related lesions. In lichen planus and lichenoid lesions, the amount of stromal tenascin paralleled the extent of inflammatory cell infiltrate in the lamina propria. In psoriform reactions and in chronic hyperplastic candidosis, the most intensive immunoreaction was seen in the connective tissue papillae between acanthotic rete ridges. Thus, the expression of tenascin seems to be associated with an enhanced epithelial cell proliferation, in line with the results of Schalkwijk et al. (1991a). A role for tenascin in epithelial-mesenchymal interplay is suggested by the marked increase in its immunoreactivity in lesions showing liquefaction degeneration. These findings are in accord with those in adult skin, which displays intense staining for tenascin around the hair follicles where a continuous epithelial- mesenchymal interaction is needed for cell proliferation (Schalkwijk et al., 1991a; Shikata et al., 1994).

There was a distinct correlation between the extent of tenascin expression and the density of the inflammatory reaction. Particularly in oral lichen planus, the sites of increased immunoreactivity coincided with the sites of most intense inflammatory reaction. Conversely, in areas devoid

of inflammation or with only a mild inflammation, tenascin expression was the same as that of normal oral mucosa. It is of interest that in lichen planus, the inflammatory cell infiltrates mostly consist of T cells (Matthews, Scully and Potts, 1984); their activation is inhibited by tenascin (Hemesath, Mar- ton and Stefansson, 1994). Thus, it may be that tenascin, in accord with its common immunomodula- tory functions (Riiegg, Chiquet-Ehrismann and Alkan, 1989; Chilosi et al., 1993) also regulates the extent of inflammatory reaction in lichen planus. Such a function has been assigned to tenascin seen, for example, in close apposition to inflammatory cells in alopecia areata (Van Baar et al., 1991) and in acne (Knaggs et al., 1994). Reciprocity in such interactions might be expected, as tenascin is induced by transforming growth factor-fl (Pearson et al., 1988), which is produced by T cells among others (Kehrl et al., 1986).

We have earlier demonstrated that enhanced expression of tenascin is frequently seen at sites with inflammatory cell infiltrates, in dysplasias and carcinomas of cervix and urinary bladder (Tiitta et al., 1992, 1993) and in inflammatory lesions of gastric and oral mucosa (Tiitta et al., 1992, 1993, 1994). Those and the present observations further empha- size the role of infection and inflammation in the regulation of the synthesis and deposition of tenascin. However, there seem to be distinct differences in the stromal response of tenascin to inflammation in different tissues, as exemplified by different types of gastric lesions (Tiitta et al,, 1994).

There is a conspicuous increase in the production of tenascin during wound healing, as shown in incision, excision and laser wounds of rat oral mucosa (Luomanen and Virtanen, 1993) and in epidermal wounds in rats (Mackie et al., 1989) and sheep (Whitby et al., 1991). In the study on sheep it was proposed that the rapid epithelialization of fetal wounds might be due to the early appearance of tenascin. A marked enhancement in the production of tenascin is also seen in gastric and duodenal ulcers and in ulcers associated with various kinds of tumours (Tiitta et al., 1994). The accentuation of its production in erosiOns and ulcers associated with oral lichen planus and lichenoid reactions seen here adds to the evidence that enhanced expression of tenascin is a general phenomenon seen in lesions involving the epithelial-stromal interface.

(Figs 5-8 Opposite)

Fig. 5. Biopsy specimen with a diagnosis of lichenoid reaction. In the epithelium, an orthokeratotic surface and slight basal-cell liquefaction degeneration are visible. A moderate mononuclear inflammatory cell infiltrate which is not strictly band-like is seen. The tenascin immunoreaction in the lamina propria is

enhanced but not as distinct as in lichen planus, x 100.

Fig. 6. In an ulcerative area of a lichenoid lesion, a marked increase in tenascin content is seen in the lamina propria, x 100.

Fig. 7. Histological diagnosis of psoriform reaction showing parakeratosis, and alternating thick and thin epithelial areas; some neutrophils are visible in the epithelium; dilated capillaries are present in the connective tissue papillae; chronic inflammatory cell infiltation is seen in the upper connective tissue. A clearly increased tenascin reaction is seen in the connective tissue papillae between the epithelial rete ridges.

x 100.

Fig. 8. Connective tissue papillae underlying the acanthotic epithelium in chronic hyperplastic candidosis display a moderate accumulation of tenascin.


Figs 5-8. Captions opposite.


Fig. 9. In control immunostaining experiments with specimens from patients with psoriform reaction (a, b) or lichen planus (c, d), the alkaline phosphatase reaction is seen as a dense precipitate when the sections are immunostained for tenascin (a, c) while only the nuclear background staining is seen in specimens in which the monoclonal antibody to tenascin has been replaced with an irrelevant antibody.

× 150.

Acknowledgements--The skilful technical and secretarial assistance of Mrs Marja-Leena Piironen, Mrs Outi Rauanheimo, Ms Marja-Liisa Tiainen and Mr Reijo Karppinen is greatfully acknowledged. The study was supported by the Finnish Medical Research Council and by the Zonta International District XX.

REFERENCES

Aufderheide E., Chiquet-Ehrismann R. and Ekblom P. (1987) Epithelial-mesenchymal interactions in the devel- oping kidney lead to expression of tenascin in the mesenchyme. J. cell Biol. 105, 599~08.

Aukhil I., Joshi P., Yan Y. and Erickson H. P. (1993) Cell- and heparin-binding domains of the hexabrachion arm identified by tenascine expression proteins. J. biol. Chem. 268, 2542-2553.

Becker J., Schuppan D. and Muller S. (1993) Immunohisto- chemical distribution of collagens type I, III, IV and VI, of undulin and of tenascin in oral fibrous hyperplasia. J. oral Pathol. Med. 22, 463-467.

Borsi L., Carnemolla B., Nicolo G., Spina B., Tanaa G. and Zardi L. (1992) Expression of different tenascin isoforms in normal, hyperplastic and neoplastic human breast tissues. Int. J. Cancer 52, 688~i92.

Chilosi M., Lestani M., Benedetti A., Montagna L., Pedron S., Scarpa A., Menestrina F., Hirohashi S., Pizzolo G. and Semenzato S. (1993) Constitutive expression of tenascin in T-dependent zones of human lymphoid tissues. Am. J. Pathol. 143, 1348 1355.

Chiquet-Ehrismann R., Mackie E. J., Pearson C. A. and Sakakura T. (1986) Tenascin: an extracellular matrix protein involved in tissue interactions during fetal devel- opment and oncogenesis. Cell 47, 131-139.

Chiquet-Ehrismann R. (1990) What distinguishes tenascin from fibronectin. FASEB J. 4, 2598-2604.

Erickson H. P. (1994) Evolution of the tenascin family-- implications for function of the C-terminal fibrinogen-like domain. Persp. dev. Neurobiol. 2, 9-19.

Erickson H. P. and Bourdon M. A. (1989) Tenascin: an extraceUular matrix protein prominent in specialized em- bryonic tissues and tumors. Annu. Rev. cell Biol. 5, 71-92.

Hemesath T. J., Marton L. S. and Stefansson K. (1994) Inhibition of T cell activation by the extracellular matrix protein tenascin. J. Immunol. 152, 5199-5207.

Howeedy A. A., Virtanen I., Laitinen L., Gould N. S., Koukoulis G. K. and Gould V. E. (1990) Differential distribution of tenascin in the normal, hyperplastic and neoplastic breast. Lab. Invest. 63, 798-806.

Jones F. J., Burgoon M. P., Hoffman S., Crossin K. L., Cunningham B. A. and Edelman G. M. (1988) A cDNA clone for cytotactin contains sequences similar to epidermal growth factor-like repeats and segments of fibronectin and fibrinogen. Proc. natn. Acad. Sci. U.S.A. 85, 2186-2190.

Kehrl J. H., Wakefield L. M., Roberts A. B., Jakowlew S., Alvarez-Noon M., Deryck R., Sporn M. B. and Fauci A. S. (1986) Production of transforming growth factor fl by human T lymphocytes and its potential role in the regulation o fT cell growth. J. exp. Med. 163, 1037-1050.

Knaggs H. E., Layton A. M., Morris C., Wood E. J., Holland B. D. and Cunliffe W. J. (1994) Investigation of the expression of the extracellular matrix glycoproteins tenascin and fibronectin during acne vulgaris. Br. J. Dermatol. 130, 576-582.

Koch M., Wehrle-Haller B., Baumgarten S., Spring J., Brubacher D. and Chiquet M. (1991) Epithelial synthesis of tenascin in at tips of growing bronchi and graded accumulation in basement membrane and mesenchyme. Exp. cell Res. 194, 297-300.

Koukoulis G. K., Gould V. E., Bhattacharyya A., Gould J. E., Howeedy A. A. and Virtanen I. (1991) Tenascin in normal, reactive, hyperplastic and neoplastic tissues: bio- logic and pathologic implications. Human Pathol. 22, 636~43.

Lightner V. A., Marks J. R. and McCachren S. (1994) Epithelial cells are an important source of tenascin in normal and malignant human breast tissue. Exp. cell Res. 210, 177-184.

Linnala A., Baiza E., Zardi L. and Virtanen I. (1993) Human amnion epithelial cells assemble tenascins and three fibronectin isoforms in the extracellular matrix. FEBS Lett. 317, 74~78.

Luomanen M. and Virtanen I. (1993) Distribution of tenascin in healing incision, excision and laser wounds. J. oral Pathol. Med. 22, 41-45.


Matthews J. B., Scully C. M. and Potts A. J. C. (1984) Oral lichen planus: an immunoperoxidase study using monoclonal antibodies to lymphocyte subsets. Br. J. Dermatol. 111, 587 595.

Mackie E. J., Halfter W. and Liverani D. (1988) Induction of tenascin in healing wounds. J. cell Biol. 107, 2757-2767.

Natali P. G., Nicotra M. R., Bigotti A., Botti C., Castellani P., Risso A. M. and Zardi L. (1991) Comparative analysis of the expression of the extracellular matrix protein tenascin in normal human fetal, adult and tumor tissues. Int. J. Cancer 47, 811-816.

Pearson C. A., Pearson D., Shibahara S., Hofsteenge J. and Chiquet-Ehrismann R. (1988) Tenascin: cDNA cloning and induction by TGF-/L E M B O J. 7, 2977-2981.

Prieto A. L., Jones F. S., Cunningham B. A., Crossin K. L. and Edelman G. M. (1990) Localization during develop- ment of alternatively spliced forms of cytotactin mRNA by in situ hybridiz~Ltion. J. cell Biol. 111, 685~98.

Riiegg C. R., Chiquet--Ehrismann R. and Alkan S. S. (1989) Tenascin, and extracellular matrix protein, exerts im- munomodulatory activities. Proc. natn. Acad. Sci. U.S.A. 86, 7437-7441.

Reichart P. A., van Wyk C. W., Becker J. and Schuppan D. (1994) Distribution of procollagen type Ill, collagen type VI and tenascin in oral submucous fibrosis (OSF). J. oral Pathol. Med. 23, 394-398.

Schalkwijk J., Steijlen P. M., Van Vlijmen-Willems I. M. J. J., Oosterling B., Mackie E. J. and Verstraeten A. A. (1991a) Tenascin expression in human dermis is related to epidermal proliferation. Am. J. Pathol. 139, 1143-1150.

Schalkwijk J., Van Vlijmen I. and Oosterling B. (1991b) Tenascin expression in hyperproliferative skin diseases. Br. J. Dermatol. 12.4, 13-20.

Shikata N., Oyaizu T., Andachi H. and Tsubura A. (1994) Tenascin expression in normal human adult skin and skin appendage tumours. Virchows Arch. 424, 511-516.

Shrestha P., Sakamotc, F., Takagi H., Yamada T. and Mori

M. (1994) Enhanced tenascin immunoreactivity in leuko- plakia and squamous cell carcinoma of the oral cavity: an immunohistochemical study. Oral Oncol., Eur. J. Cancer 30B, 132-137.

Siri A., Carnemolla B., Saginati M., Leprini A., Casari G., Baralle F. and Zardi L. (1991) Human tenascin: primary structure, pre-mRNA splicing patterns and localization of the epitopes recognized by two monoclonal antibodies. Nucl. acid Res. 19, 525-531.

Sloan P., Schor S. L., Lopes V. and Chiquet-Ehrismann R. (1990) Immunohistochemical study of the heterogeneity of tenascin distribution within the oral mucosa of the mouse. Archs oral Biol. 35, 67-70.

Soini Y., Lehto V.-P. and Virtanen I. (1993) Tenascin in human lung tumours. Am. J. Clin. Pathol. 100, 145 150.

Tiitta O., Wahlstr6m T., Paavonen J., Linnala A., Sharma S., Gould V. E. and Virtanen I. (1992) Enhanced tenascin expression in cervical and vulvar koilocytotic lesions. Am. J. Pathol. 141, 907-913.

Tiitta O., Wahlstr6m T., Virtanen I. and Gould V. E. (1993) Tenascin in inflammatory conditions and neoplasms of the urinary bladder. Virchows Arch. 63, 283-287.

Tiitta O., Happonen R. P., Virtanen I. and Luomanen M. (1994) Distribution of tenascin in oral premalignant lesions and squamous cell carcinoma. J. oral Pathol. Med. 23, 446-450.

Tiitta O., Sipponen P., Gould V. and Virtanen I. (1994) Tenascin expression in inflammatory, dysplastic and neoplastic lesions of human stomach. Virchows Arch. 425, 369 374.

Van Baar H. M. J., Perret C. M., van Vlijmen-Willems I. M., Cleutjens J. P., Mackie E. J., Happle R. and Schalkwijk J. (1991) Expression of tenascin in perifollicu- lar connective tissue: comparison of normal scalp and alopecia areata. Arch. Derrnatol. Res. 283, 86-90.

Whitby D. J., Longaker M. T., Harrison M. R., Adzick N. S. and Ferguson M. W. J. (1991) Rapid epithelialisa- tion of fetal wounds is associated with the early deposition of tenascin. J. cell Sei. 99, 583-586.

Documents

Tenascin expression in mucocutaneous diseases and related lesions of human oral mucosa