IMMUNO-HISTOCHEMISTRY NORMAL AND DISEASED EPITHELIUM L. K.

OBERLE,

OF KERATIN IN HUMAN ORAL

K. FUKUYAMA,T. E. DANIELS and J. S. GREENSPAN*

Department of Oral MedicineiHospital Dentistry, School of Dentistry, Department of Dermatology. School of Medicine. University of California. San Francisco, CA 94143. U.S.A.

Summary-Immuno-reactivity of human oral epithelial proteins was investigated by indirect immuno-fluorescence microscopy in normal and pathological conditions. Reactivity to antiserum was seen throughout the epithelium in normal oral mucosa and in lichen planus. Reactivity of epithelial cells was variable in erythema multiforme, pemphigoid and pemphigus.

IKTRODUCTIOK

Shimizu. Fukuyama and Epstein (1974) isolated a keratin consisting of two polypeptides from cornified cells of newborn rat skin and raised antisera to this protein in the rabbit. This rabbit anti-rat serum (RARS) cross-reacts with the skin of other mammals, including man (Inoue. Fukuyama and Epstein, 1976) but not with other fibrous proteins, including actin and myosin (Fukuyama, Murozuka and Epstein, 1978). Although human oral epithelium ranges from nonkeratinized to orthokeratinized, all normal epithelial cells contain tonofilaments. Lesions of oral lichen planus show an increase in tonofilaments and bundle formation (El-Labban, 1970; Hashimoto, 1976). in contrast to those of erythema multiforme (Von Billow, Hjwting-Hansen and Ulmansky, 1966), pemphigoid (Shklar, Meyer and Zacarian, 1969; Susi and Shklar, 1971), and pemphigus (Wilgram, Caulfield and Lever, 1961; Hashimoto and Lever. 1967, 1970). Our aim was to determine, by indirect immunofluorescence microscopy, if keratin of human oral mucosa is immunologically reactive to RARS.

MATERIALS

AW

METHODS

formaldehyde for 2 mitt, washed in two changes of phosphate-buffered saline (PBS) for 15 min, rinsed with distilled water, and air-dried. Sections were incubated with RARS (Inoue et al., 1976) for 30 min at room temperature. Control sections were incubated with normal non-treated rabbit sera under the same conditions. RARS and control sera were absorbed with rat dermal and liver proteins cross-linked with glutaraldehyde (Avrameas and Ternyneck, 1969). Tenfold dilutions (from I :20 to 1:70) of the sera in PBS were tested on normal oral mucosa to establish optimum staining conditions. The sections were washed in PBS. rinsed, then incubated in the dark for 30min with a 1: 10 dilution of fluoresceinconjugated goat anti-rabbit IgG (Hyland Co., Costa Mesa CA., F/P molar ratio, 2.8). After a further wash in the dark, the sections were air dried and mounted in glycerine. All specimens were examined with a Zeiss transmission fluorescence microscope with a high-pressure mercury vapour lamp (HBO-200). equipped with a BG 12 exciter filter and a 50 barrier filter. Newborn-rat skin was used as a positive control and rat liver and spleen as negative controls. Blocking experiments were performed by incubating tissue with non-conjugated goat anti-rabbit serum after incubation with RARS and before staining with fluorescein-labelled goat antirabbit IgG.

Ten specimens of normal mucosa were obtained from patients by oral surgical procedures yielding mucosa. Specimens of oral mucosa from five subjects with lichen planus, six with erythema multiforme, and one each with mucous membrane pemphigoid and pemphigus were additionally reacted with RARS. The specimens were quick-frozen and stored in liquid nitrogen. Cryostat sections 4 pm thick were cut at -20°C and picked up on slides coated with formol-gelatin. The sections were fixed in 4 per cent para-

RESULTS

Normal

* Requests for reprints should be addressed to: Dr. J. S. Greenspan. Department of Oral Medicine and Hospital Dentistry, 653s. University of California, San Francisco, California 94143, U.S.A. O.R 24,,5

A

oral ntucosa

Staining for keratin using the RARS antiserum occurred in all layers of the oral epithelium (Figs. 1 and 2). showing that the distribution of the protein in oral mucosa is similar to that in human and rat skin (Inoue et al., 1976). There was no relationship between the degree of keratinization and the intensity of staining. The RARS was optimally reactive at dilutions of 1:4&1:60. At higher concentrations, the reactivity of the stratum corneum of keratinized epithelium decreased. Tissue first incubated with normal rabbit sera did not show fluorescence. With optimum dilutions of RARS, the intensity of 321

L. K. Oberle, K. Fukuyama, T. E. Daniels and J. S. Greenspan

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immuno-reactivity was even throughout the epithelium. There was no reaction in nuclei, keratohyalin granules (where present), or the underlying connective with non-conjugated anti-rabbit tissue. Blocking serum greatly decreased the staining of fluorescein isothiocyanate (FITC)-conjugated antiserum. Lichen

planus

The epithelium was from the gingiva and cheek and showed hyper-ortho-keratosis and hyper-keratosis (Fig. 3A). There was a moderate infiltration of lymphocytes at the epithelialconnective tissue junction. Immuno-reactivity in the epithelium was similar to that in normal oral mucosa and was detected throughout all layers (Figs. 3B, C). The effect of serial dilutions of RARS on the stratum corneum was similar to that seen in normal oral mucosa. Erythemu

multiforme

The tissue specimens from the cheek were histologically consistent with erythema multiforme. Oedema of the prickle cell layer and some intra-epithelial vesicles were present (Fig. 4A). Immuno-reactivity was detected in all layers of the epithelium (Fig. 4B), but varied in intensity in individual cells. There was a marked reduction in staining in the lower strata, and the basal cell layer was non-reactive. There were some focal areas of reactivity within the connective tissue. Pemphigoid

The tissue was from the gingiva and showed typical subepithelial clefting (Fig. SA). Variable immunoreactivity of cells occurred throughout the epithelium (Fig. 5B). The basal cells and groups of cells nearby showed reduced staining. Pemphigus

The tissue examined was also from the gingiva and showed-moderate hyper-keratosis and characteristic supra-basal clefting with vesicle formation (Fig. 6A). Most cells, including many of the acantholytic cells, showed immuno-reactivity (Fig. 6B). However, basal cells showed low reactivity or none, in contrast with acantholytic cells which when present were highly reactive. DISCUSSION

Our results indicate that keratin of oral epithelial cells shares immunological reactivity with human skin, independent of the degree of keratinization. As the antiserum was produced by the injection of an epidermal fibrous protein, shown to be keratin, we assume that the antigenic protein is a constituent of tonofilaments. Ultrastructural studies show that the tonofilaments of keratinized epithelium are more tightly packed as the cells migrate to the surface, whereas tonofilaments in non-keratinizing cells remain in random distribution (Chen and Meyer, 1971). Differences in immuno-reactivity between antisera to epithelial keratin have been observed. Some antisera did not react with keratinized cells even though the antigenic proteins were isolated from keratinized cells (Tezuka and Freedburg, 1972; Lee et al., 1976). These differences were explained as structural or

chemical alteration of the antigenic sites during differentiation and migration of epithelial cells into the keratinized surface layer. With our antisera, dilution influenced immuno-reactivity. If the concentration of the antiserum was too high, there was less immunoreactivity in the stratum corneum, suggesting that excess antibody may also interfere with the reaction. Reactivity was variable in pathological lesions. In lichen planus, the reactivity was similar in intensity and distribution to that seen in normal keratinized epithelium. This corroborates ultrastructural findings (El-Labban, 1970) of increased tonofilaments and bundle formation in lichen planus similar to that in superficial layers of normal keratinizing oral mucosa. El-Labban (1970) and Hashimoto (1976) suggested that epithelial cells in lichen planus show premature keratinization, i.e. loss of intercellular organelles and desmosomal attachments. In erythema multiforme, changes are generally restricted to the prickle cell layers (Von Biilow et al., 1966) probably beginning as intercellular oedema, followed by intracellular changes leading to complete disruption of single cells and the basement membrane. Reduction of immuno-reactivity in our study was consistent with the location of degeneration. As the lesion in mucous membrane pemphigoid is at the epithelialconnective tissue junction (Shklar et al., 1969; Susi and Shklar, 1971) we expected reduction in immuno-reactivity to be mainly in the basal cells. In pemphigoid, the epithelium remains little changed until the bulla has been present for some time. The epithelium then becomes thin and atrophic, with some cellular degeneration in the stratum spinosum (Susi and Shklar, 1971). These findings could explain the variable immuno-reactivity of the epithehum in our study. In ultrastructural studies of pemphigus, the attachment plaques and associated tonofilaments are the last elements to break down (Hashimoto and Lever, 1967, 1970); in contrast, at the basal cell layer, the intercellular substance is preserved, and condensation of tonofilaments and their separation from the desmosomes occur early (Wilgram et al., 1961; Hashimoto and Lever, 1967). These observations may explain the finding that immuno-reactivity remains in many acantholytic cells but not in basal cells. Hersh et al. (1977) used RARS to investigate the changes associated with carcinoma induced by U.V. light in the hairless mouse. As there were no obvious changes in ultrastructure of individual tonofilaments during malignant transformation, Hersh et al. suggested the following explanations for the tumour cells’ lost immuno-reactivity : (1) absence or reduction of fibrous protein in those cells; (2) chemical changes in proteins which modify or delete the antigenic sites (3) masking of the antigenic sites. The variability of immuno-reaction in our study may be due to one or all of these. The alteration of immuno-reactivity in disease may result from the underlying mechanism, such as toxic reaction, inflammation or autoimmunity. Antigenic changes in tonofilaments may also be a primary pathological event. Continued study of the immunoreactivity of this intracellular antigen, and particularly of its variability in pathological conditions, may provide greater understanding of the cause of these diseases.

Keratin in human oral epithelium Acknowledgements-We acknowledge the skilled technical assistance of Carmen Quadra-White. This study was sup ported in part by USPHS Grant AM12433 from the National Institutes of Health, Bethesda, Maryland.

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Hashimoto K. and Lever W. F. 1970. An ultrastructural study of cell junctions in pemphigus vulgaris. Archs Derm. 101, 287-298.

Hersh L., Fukuyama K., Inoue N. and Epstein J. H. 1977. Immunofluorescent studies of epidermal protein during WV induced carcinogenesis. Virchows Arch. B Cell Path. 24, 157-164.

Inoue N., Fukuyama K. and Epstein W. L. 1976. Tmmunochemical studies of proteins in epidermal cornified cells of human and newborn rat. Biochim. bjophys. Aeta 439,

REFERENCES

Avrameas S. and Ternyneck T. 1969. The cross-linking of proteins with glu~draldehyde and its use for the preparation of immunoadsorbents. immunochemistry 6, 53-66. Chen S. Y. and Meyer J. 1971. Regional differences in tonofilaments and keratohyalin granules. In: Current Concepts of the Histology of Oral Mucosa (Edited by Squier C. A. and Meyer J.) pp. 114128. Charles C. Thomas, Springfield, Ill. El-Labban N. G. 1970. Light and electron microscopic studies of colloid bodies in lichen planus. J. periodont. Res. 5, 31.5-324. Fukuvama K.. Murozuka T., Caldwell R. and &stein W.-L. 1978. Divalent cation stimulation of an in’ vitro fibre assembly from epidermal keratin protein. 1. Cell. Sci. 33, 255-263. Hashimoto K. 1976. Apoptosis in lichen planus and several other dermatoses. Acta derm.-uener. 56, 187-210. Hashimoto K. and Lever W. F. 1967. An electron microscopic study on pemphigus vulgaris of the mouth and the skin with special reference to the intercellular cement. .I. invest. Derm. 48, 540-552.

95-106.

Lee L. D., Baden H. P., Kubilus J. and Fleming B. F. 1976. Immunology of epidermal fibrous proteins. J. inuest. Derm. 67, 521-525. Shim& T., Fukuyama K. and Epstein W. L. 1974. Partial purification of proteins isolateci from mammalian cornified cells. Biochim. biophvs. Acta 359, 389400. Shklar G., Meyer 1. anh iacarian S. 1969. Oral lesions in bullous pemphigoid. Archs Derm. 99, 663-670. Susi F. R. and Shklar G. 1971. Histochemistry and fine structure of oral lesions of mucous membrane pemphigoid. Archs Derm. 104, 244-253. Tezuka T. and Freedburg I. M. 1972. Epidermal structural proteins. II. Isolation and purification of tonofilaments of the newborn rat. Biochim. biophys. Acta 263, 382-396. Von BulGw F. A., Hj0rting-Hansen E. and Ultnansky M. 1966. An elcc~onmicroscopic study of oral mucosal lesions in erythema multiforme exudativum. Acta path. microbioi. stand. 66, 145-153. Wiigram G. F., Caulfield J. B. and Lever W. F. 1961. An electron microscopic study of acantholysis in pemphigus vulgaris. J. invest. Derm. 36, 373-382.

Plates 1 and 2 overieaf.

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L. K. Oberle, K. Fukuyama, T. E. Daniels and J. S. Greenspan

Plate 1. Figs. 1 and 2. Normal buccal mucosa (Fig. 1) and gingiva (Fig. 2) 4-pm cryostat sections. (A) Stained with hematoxylin and eosin. (B). Sections incubated with RARS followed by fluorescein-conjugated goat anti-rabbit IgG. Immuno-reactivity is present in all layers of the epithelium regardless of the degree of keratinization. (C) Control sections incubated with normal rabbit sera followed by fluoresceinconjugated goat anti-rabbit IgG. No immuno-reactivity. x 300 Fig. 3. Lichen planus. 4pm cryostat sections of gingiva. (A) Stained with hematoxylin and eosin. The euithelium exhibits hvuer-ortho-keratosis. (B) Section incubated with RARS followed by fluorescein-. conjugated goat anti-rabbit IgG. Immuno-reactivity is present throughout all layers. (C) Control section incubated with normal rabbit sera followed by fluorescein-conjugated goat anti-rabbit IgG. No immuno-reactivity. x 300 Plate 2. Fig. 4. Erythema multiforme. 4pm cryostat sections of buccal mucosa. (A) Stained with hematoxylin and eosin. Oedema of the prickle cell layer and some intra-epithelial vesicles are present. (B) Section incubated with RARS followed by fluorescein-conjugated goat anti-rabbit IgG. Reduction in staining is more pronounced in the lower strata (arrow). x 300 Fig. 5. Pemphigoid. 4 pm cryostat sections of gingiva. (A) Stained with hematoxylin and eosin showing typical subepithelial clefting. (B) Section incubated with RARS followed by fluorescein-conjugated goat anti-rabbit IgG. Reduced staining is present in the basal cells (arrow) and groups of cells nearby. x300 Fig. 6. Pemphigus. 4 pm cryostat sections of gingiva. (A) Stained with hematoxylin and eosin, showing hyperkeratosis and characteristic supra-basal clefting. (B) Section incubated with RARS followed by tluorescein-conjugated goat anti-rabbit IgG. Immune-reactivity is present in most cells, including many acantholytic cells. Basal cells (arrow) show low reactivity or none. x 300

Keratin in human oral epithelium

Pfate 1.

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L. K. Oberle, K. Fukuyama,

T. E. Daniels and J. S. Greenspan

Plate 2.

Immuno-histochemistry of keratin in normal and diseased human oral epithelium.

IMMUNO-HISTOCHEMISTRY NORMAL AND DISEASED EPITHELIUM L. K. OBERLE, OF KERATIN IN HUMAN ORAL K. FUKUYAMA,T. E. DANIELS and J. S. GREENSPAN* Departm...
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