Ultrastructure of eosinophilic bodies in the degenerative surface epithelium of chronic hyperplastic oral lesions

Ultrastructure of eosinophilic bodies in the degenerative surface epithelium of chronic hyperplastic oral lesions Sow-Yeh Chen, Ph.D., Philadelphia,

Pa.

DEPARTMENT

UNIVERSITY

OF PATHOLOGY,

TEMPLE

SCHOOL

OF DENTISTRY

Eosinophilic bodies were located extracellularly in the dilated intercellular spaces of degenerative epithelial cells. They consisted of electron-dense amorphous material with many scattered mature fibrin fibers. Eosinophilio bodies were PAS positive, mucicarmine negative, aleian blue negative. A large number of lysosome-like bodies with homogeneous electron-dense content were present in the cytoplasm of the surrounding degenerative epithelial cells. The plasma membrane of these cells was intact and there was no outpouring of tonofilaments or keratohyaline granules into the intercellular spaces. With the results of the present study, it is suggested that eosinophilic bodies are probably an exudate of plasma fluid accumulated in the dilated intercellular spaces of superficial degenerative cells.

E

osinophilic bodies in the superficial epithelial cell layers of oral inflammatory hyperplastic lesions have been reported by many investigators.1-4 Toto described the eosinophilic bodies as homogeneous masses conforming in size and shape to the superficial prickle cells and referred to such change in the superficial epithelium as “mucopolysaccharide keratin dystrophy.” Archard and Glass2 found similar changes in epulis fissuratum and irritation fibromas and referred to the process by which the eosinophilic material forms as “keratin pooling.” Cutright? found homogeneous eosinophilic vesiculation in 19 of 583 cases of epulis fissuratum. The eosinophilic vesiculation was in the very upper layers of the stratum spinosum and even in the parakeratinized layers. Buchner, Mlinek, and CalderorP observed eosinophilic bodies in 53 of 423 oral inflammatory hyperplastic lesions. They referred to these eosinophilic bodies as “keratin-like material.” The present study was undertaken to investigate the ultrastructural features of the eosinophilic bodies. This study was supported by Grant No. IN-88F from the American Cancer Society, a grant-in-aid from Temple University, and Grant No. T1208144-08 from the National Cancer Institute, National Institutes of Health, Bethesda, Md.

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Pig. 1. Eosinophilic bodies in the superficial portion of an epithelium associated with pyogenic granuloma. (Hematoxylin and eosin stain. Magnification, x125.) Fig. d. Eosinophilic bodies in the superficial portion of an epithelium associated with inflammatory fibrous hyperplasia. (Hematoxylin and eosin stain. Magnification, x125.)

MATERIALS AND METHODS Two specimens of pyogenic granuloma and three of inflammatory fibrous hyperplasia were used. Specimens were fixed in phosphate-buffered 10 per cent formalin for ‘72 hours. After washing overnight with 0.08M cacodylate buffer of pH 7.2, specimens were minced into small cubes and postfixed with 1 per cent osmium tetroxide in 0.05M cacodylate buffer of pH 7.2, dehydrated through ascending concentrations of ethanol and acetone, then embedded in Epon 812.5 Thin sections of 600 to 800 ii were stained with uranyl acetate6 and lead citrate,7 and examined with a Philips EM 300 electron microscope. Paraffin sections of each specimen were treated with McManus’ periodic acid-Schiff (PAS) method controlled with diastase,s Mayer’s mucicarmine,8 alcian blue,s and Mallory’s phosphotungstic acid hematoxylin (PTAH) methods for fibrin.

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C’hez

Oral Hurg. February, 19i7

Pig. 3. Electron micrograph of dilated intercellular spaces of variable size. (Magnification, x11,200.) Eosinophilic bodies appear to be dilated intercellular spaces filled with amorphous electron-dense material. Some fibrin fibers (thin arrows) and circular lucid areas are scattered in the amorphous material. Lysosome-like bodies (thick arrows) are present in the surrounding degenerative epithelial cells.

RESULTS

As described by other investigators,l+ ovoid or roughly round eosinophilic bodies were most often present in the superficial prickle-cell layers and/or parakeratin layer (Figs. 1 and 2). Vacuolation of the eosinophilic bodies was commonly observed. The eosinophilic bodies were separated from each other by a

Eosinophilic

Fig. 4. Electron micrograph like projections. (Magnification,

of dilated x11,200.)

bodies in degenerative

intercellular

spaces surrounded

epithelium

259

by cells with villus-

thin band of cytoplasm. A pyknotic nucleus was often associated with the thin band of cytoplasm. The corresponding underlying lamina propria was edematous and chronically inflamed. By means of electron microscopy, eosinophilic bodies were located extracellularly in dilated intercellular spaces (Fig. 3). They were composed of electrondense amorphous material, Similar to what was observed under the light microscope, these dilated spaces were separated from each other by a thin band of

260

Cheer

Fig. 5. Electron micrograph of a large distended intercellular dense amorphous material with many fibrin fibers. (Magnification, cular lucid areas are scattered in the amorphous material.

Oral surg. Fc~l,runry, 197;

space containing x11,200.) Many

electrow small cir-

cytoplasmic processes of adjacent cells. The cytoplasmic processes were joined to each other by desmosomes. Frequently a whole cell was compressed to become a thin band of cytoplasm with a pyknotic nucleus adjacent to dilated intercellular spaces. The plasma membrane of epithelial cells surrounding the spaces was intact and appeared smooth or corrugated, and some plasma membrane even showed villus-like projections (Fig. 4). In the electron-dense amorphous material, there

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Fig. 6. High-magnification electron fibers. (Magnification, x37,500.)

bodies ilz degenerative

micrograph

showing

cross-striations

epithelium

of mature

261

fibrin

were scattered fibrin fibers of variable size (Figs. 3 to 5). The longest dimension of the fibers observed was 3,1. and the widest dimension was 0.5~. At higher magnification, these fibrin fibers showed cross-striations along their longitudinal axes (Figs. 6 and 7). The cross-striations were characterized by dark bands of 120 ,% thickness alternating with light bands of 70 A thickness. Minute lucid vacuoles not limited by a membrane were also scattered in the amorphous material (Figs. 3, 4, 5, and 8). In addition, fine filaments of approximately 75 A thickness, probably primitive fibrin fibrils, were present in small focal areas of some spaces (Fig. 8). Epithelial cells surrounding the dilated intercellular spaces revealed degenerative changes (Figs. 3, 9, and 10). Small ovoid or irregular lucid areas were scattered among tonofilaments in the cytoplasm. Some dilated profiles of rough-surfaced endoplasmic reticulum and mitochondria with disrupted cristae were also present in the cytoplasm (Figs. 9 and 10). Lysosome-like bodies with homogeneous electron-dense content were present in most cells. These bodies were scattered predominantly in the peripheral portion of cytoplasm (Figs. 3, 4, 9, and 10). Fusion of lysosome-like bodies with plasma membrane was occasionally observed (Fig. 10). Despite the degenerative changes in the cytoplasm and the presence of lysosome-like bodies, the plasma membrane was intact. In parakeratinized cells and many superficial epithelial cells, the plasma membrane was thickened at the inner leaflet. Outpouring of tonofilaments into the intercellular spaces was not observed. No keratohyaline granules were observed. A moderate

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Chen

OELl Hurg. February, 19i7

Fig. 7. Electron micrograph of fibrin fibers with cross-striations in small intercellular spaces. (Magnification, x25,000.) Intercellular spaces are filled with electron-dense amorphous material. Glycogen particles are scattered in the cytoplasm of surrounding cells.

number of glycogen particles were scattered in the cytoplasm of degenerative cells (Fig. 7). With histochemical procedures, eosinophilic bodies were PAS positive and diastase resistant, indicating the presence of carbohydrates (Table I). However, they were mucicarmine negative and alcian blue negative, indicating the absence of mucin. Eosinophilic bodies appeared blue with PTAH stain, suggesting the presence of fibrin. The plasma fluid in the dilated vascular channels of the underlying lamina propria was eosinophilic and PAS positive, and also stained blue with Mallory’s PTAH method. DISCUSSION The present study demonstrated that eosinophilic bodies in the superficial epithelial cell layers accumulate within dilated intercellular spaces. These eosinophilic bodies are not intracellular bodies or pooling of keratin as considered previously by Toto and by Archard and Glass.* The plasma membrane of degenerative epithelial cells surrounding the spaces was intact and there was no

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Fig. 8. Electron micrograph of small areas composed of thin filaments (arrows) in dilated intercellular spaces. (Magnification, x16,800.)

Table

I. Histochemical

findings of eosinophilic

Component

Present or absent

Protein

Present

Arginine Sulfhydryl group

Absent Present

Disultide Tryptophan

Present Present

Carbohydrate

Present

Acid mucopolysaccharide

Keratin

material

Present Absent Present

I

Reference

: 2 i 4 : I i Present study 1 : Present study I

outpouring of keratin. Eosinophilic bodies were located extracellularly and composed of electron-dense amorphous material with scattered mature fibrin fibers and occasional fine filaments. The amorphous material is similar to coagulated blood plasma, and the fibrin fibers are similar to those described elsewhere.9* lo Eosinophilic bodies appear to result from pooling of plasma fluid in the intercellular spaces. It has also been suggested previously by Buchner, Mlinek, and Calderon4 that

264

Chen

Pig. 9. Electron micrograph of a degenerative cell adjacent to dilated intercellular (Magnification, x11,200.) Lysosome-like bodies and cytoplasmatic vacuolated areas present.

spaces. (V) are

the eosinophilic material might represent a filtrate from the blood vessels. The presence of eosinophilic material in the epithelium occurs only above areas showing an inflammatory process. The incidence of occurrence is closely related to the severity of inflammation. Among oral inflammatory lesions, the incidence is highest in pyogenic granuloma.4 The results of the present study support the idea of Buchner and associates4 that eosinophilic material in the superficial

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Fig. 10. Electron micrograph of approach (arrows). (Magnification, x11,200.)

bodies ill degenerative

of lysosome-like

bodies

epithelium

to plasma

265

membrane

epithelial cell layers is an accumulation of blood plasma permeated from the underlying inflamed connective tissue and is, in most aspects, similar to inflammatory exudate. The most common locations for accumulation of eosinophilic material are the superficial epithelial cell layers and parakeratin layer.lm4 The superficial cell layers of inflamed oral mucosa underwent degenerative changes. Lysosome-like bodies were present in most cells surrounding the pools of eosinophilic material. Unlike membrane-coating granules, I1 lysosome-like bodies did not have highly ordered internal lamellae. They contained homogeneous electron-dense material. Similar lysosome-like bodies have been observed by Listgarten12 in chronically inflamed gingiva. These superficial cells are apparently weakened by degenerative changes and become compressible. An extremely compressed, thin cell with a pyknotic nucleus around a pool of eosinophilic material was often observed in the present study. Besides cytoplasmic degeneration, the plasma membrane of superficial epithelial cells and parakeratinized cells was thickened. A thickened plasma membrane is less permeable to macromolecules such as fibrin. Thus, it is reasonable for fibrin and other plasma components to accumulate in the intercellular spaces. The eosinophilic material is composed of protein and carbohydrate.‘, 2l 4 It is

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Chen

PAS positive and considered to be partially composed of carbohydrate.lx p, a Glycoproteins and mucoproteins are normal components of blood plasma.‘” Most of the protein and carbohydrate in the eosinophilic material may represent those derived from the permeated plasma. In the present observation, plasma fluid in vascular channels was indeed PAS positive. Furthermore, fibrin itself consists of protein and carbohydrates, particularly sialic acid.” Possibly glycoproteins and mucopolysaccharides of normal intercellular substancel” are mixed with accumulated plasma components ; however, the contribution of normal intercellular substance to the formation of eosinophilic bodies is very small as the whole volume of eosinophilic bodies is concerned. The author thanks Mrs. Pamela Conway, Mr. Richard Geissler, and Mrs. Regina for technical assist,anee, and Ms. Kathy Ravert for typing the manuscript.

Tyranski

REFERENCES

Keratin Dystrophy of the Oral Epithelium, 1. Toto, P. D.: Mucopolysaccharide ORAL SURG. 22: 47-48, 1966. 2. Archard, H. O., and Glass, N. M.: Degenerative Changes in the Superficial Epithelium of and Histochemical Study, J. Dent. Chronic Hyperplastic Oral Mueosa : Clinicopathological Res. 49: 1118-1124, 1970. ORAL 3. Cutright, D. E.: The Histopathologie Findings in 583 Cases of Epulis Fissuratum,

SURQ.37: 401-411.1974. Bodies in the Epithelium of Oral 4. Buchner, A., Mlinek, A., and Calderon, S.: Eosinophilic Inflammatory Hyperplastic Lesions, ORAL SURQ.41: 378-384, 1976. 5. Luft, J. II.: Improvements in Epoxy Resin Embedding Methods, J. Biophys. Biochem. Cytol. 9: 409-414, 1961. 6. Watson, M. L.: Staining of Tissue Sections for Electron Microscopy with Heavy Metals, J. Biophys. Biochem. Cytol. 4: 475-478, 1958. 7. Reynolds, E. S.: The Use of Lead Chtrate at High pH as Electron-Opaque Stain in Electron Microscopy, J. Cell Biol. 17: 208-212, 1963. 8. Luna, L. G.: Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology, ed. 3, New York, 1968, The Blakiston Division, McGraw-Hill Book Company, Inc. 9. Pouit, L., Hudry-Clergeon, Study of Positive G., and Suseillon, M.: Electron Microscopy Changes on Fibrin Fibers, Biochim. Biophys. Acta 317: 99-105, 1973. 10. Conio, ct., Dondero, G., Troglia, C., Trefiletti, V., and Patrone, E.: The Structure of Fibrin. An Electron Microscopic Investigation, Biopolymers 14: 2363-2372, 1975. 11. Lavker, R. M. : Membrane Coating Granules: The Fate of the Discharged Lamellae, J. Ultrastruct. Res. 55: 79-86, 1976. 12. Listgarten, M. A.: Unusual Organelle in Surface Cells of Inflamed Gingival Epithelium, Abst. No. 38, p. 58, The 50th General Session of the International Association for Dental Research, Las Vegas, Nevada, 1972. 13. Orten, J. M., and Neuhaus, 0. W.: Human Biochemistry, ed. 19, St. Louis, 1975, The C. V. Mosby Company, chap. 17, p. 751. 14. White, A., Handler, P:, and Smith, E. L.: Principles of Biochemistry, ed. 5, New York, 1973, The Blakiston Division, McGraw-Hill Book Company, Inc., chap. 30, pp. 820-824. 15. I bal, M., and Gerson, 8.: Biochemical Features of Oral Epithelium. In S uier, C. A., and Ill., It!?eyer, J., editors: Current Concepts of the Histology of Oral Mucosa, 1 pringfield, 1971, Charles C Thomas, Publisher, chap. 2, p. 48. Reprint requests to: Dr. Sow-Yeh Chen Temple University School of Department of Pathology 3223 North Broad St. Philadelphia, Pa. 19140

Dentistry