INTRAEPITHELIAL MAST CELLS IN GINGIVAL LICHEN PLANUS: AN ULTRASTRUCTURAL STUDY

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1 THE JOURNAL OF INVESTIGATIVE DERMATOLOTY, 64: , 1975 Copyright 1975 by The Williams & Wilkins Co. Vol. 64, No.6 Printed in U.S.A. INTRAEPITHELIAL MAST CELLS IN GINGIVAL LICHEN PLANUS: AN ULTRASTRUCTURAL STUDY MICHAEL L. BARNETT, D.D.S. Department of Periodontics, School of Dentistry, State University of New York at Buffalo, Buffalo, New York Intraepithelial mast cells, identified by ultrastructural criteria, were seen in lesions of gingival lichen planus. The mast cells were found either singly, interspersed among keratinocytes, or in combination with other mononuclear cell types, especially lymphocytes. The mast cells were seen in regions with relatively normal intercellular spaces, as well as in regions of more severely disrupted keratinocytes. They had cytologic features indicative of active synthesis and release of granules. Moreover, the finding of centrioles in several intraepithelial mast cells, in combination with certain other cytologic features, suggested that these cells might be in an early stage of differentiation. It is speculated that intraepithelial mast cells have a role in the pathogenesis of gingival lichen planus. Mast cells have been classically considered as connective tissue elements which, by virtue of their content of biologically active substances such as histamine, heparin, and proteolytic enzymes, may have a role in the pathogenesis of certain tissue responses [1-3]. Previous light microscopic studies have suggested that mast cells may also occasionally be found in intraepithelial sites [3-5 ]. Recently, this has been confirmed by electron microscopy which has dem.onstrated that mast cells are present within a variety of epithelia, including normal [6] and inflamed [7] human gingival epithelium, diseased human renal epithelium [8 ], human epidermis affected with alopecia mucinosa (9], and rat intestinal epithelium in the course of parasite expulsion [10,11]. In the case of normal gingival oral epithelium, for example, it appears as though mast cells emigrate across the basal lamina from lamina propria to epithelium and subsequently become separated from the lamina propria by intervening keratinocyte processes and the restoration of the basal lamina [6]. Since considerations of the pathogenesis of specific diseases include the types of cells participating as well as the sequence in which they participate, a study was undertaken to identify, using ultrastructural criteria, the cell types included in the so-called chronic inflammatory infiltrate [12] of gingival lichen planus, with special emphasis on that portion of the infiltrate found within the epithelium. In this report evidence is presented to show that mast cells are included in this intraepithelial cell population and, moreover, that they Manuscript received December 12, 1974; in revised form January 27, 1975; accepted for publication January 28, Supported by USPHS Grants DE and DE from the National Institute for Dental Research. Reprint requests to: Dr. M. L. Barnett, Department of Periodontics- Endodontics, School of Dentistry, SUNY at Buffalo, Buffalo, New York have ultrastructural features consistent with active synthesis and release of granule components. In addition, because a small number of these intraepithelial mast cells appeared similar to mast cells in early stages of differentiation [13-15], the Implications with respect to their derivation are aiscussed. MATERIALS AND METHODS Specimens of buccal gingiva with lesions of lichen planus were obtained by sharp dissection and minimal tissue manipulation from 3 female patients, aged 48, 55, and 57. Clinically, the lesions appeared either as erosive lichen planus (2 cases) or as a " hyperkeratotic' 1 form (2 cases), with 1 patient having both forms in different sites. The diagnosis of lichen planus was confirmed by conventional histopathologic methods. For ultrastructural study, tissue specimens from each patient were fixed for 2 hr in a phosphate-buffered modified Karnovsky fixative [16], ph 7.4, at room temperature, postfixed for 1 hr in 1% osmium tetroxide, and dehydrated and embedded in Epon utilizing standard methodology. Thick sections were stained with toluidine blue for surveying of the tissue. Portions of the tissue with obvious histopathology were thin sectioned. The thin sections were stained with uranyl acetate and lead citrate [i 7 ], and photographed with either an AEI EM6-B or an Hitachi HS-8 electron microscope. RESULTS lntraepithelial mast cells were observed in all the specimens examined. The mast cells were identified by virtue of well-established ultrastructural criteria [18], including substructure of specific granules, presence of surface microvilli, and morphology of the nucleus. These cells were located primarily in the basal and spinous layers of the epithelium and were found either singly, interspersed among keratinocytes (Figs. 1, 2), or in combination with other mononuclear cells (Fig. 3). The mononuclear cell types present included lymphocytes, macrophages, and Langerhans cells. Occasionally, a mast cell had a form which suggested

2 June 1975 INTRAEPITHELIAL MAST CELLS IN LICHEN PLANUS 437 FJG. 1. Intraepithelial mast cell, lichen planus. The mast cell (me) has surface microvilli and granules with substructures. The nucleus contains a prominent nucleolus. Mitocho.n dria are arranged in a perinuclear zone. A very prominent Golgi zone (g) and centrioles (c) are present in the region of nuclear indentation. The granules are rather sparsely distributed throughout the cell; in addition, empty vacuoles (arrows) near the cell periphery are suggestive of partial degranulation. Also evident are free ribosomes, profiles of rough endoplasmic reticulum, microfilaments, and microtubules. No intercellular junctions are seen between the mast cell and adjacent keratinocytes (k), although the cells are in close apposition to each other. The Golgi region of the mast cell is shown at higher magnification in Fig. 4 ( x 10,400). char::tdpri~t.ic that it might be migrating through the tissue. In addition, bizarre configurations of mast cells, consistent with large surface concavities, were sometimes seen. Although mast cells were often in close apposition to keratinocytes or other mononuclear cells, no specialized cell junctions were formed between the mast cell and these other cell types (Figs. 1-3). It did not appear as though severe epithelial disruption was a necessary precursor for mononuclear cell infiltration of the epithelium, since mast cells and other mononuclear cells were seen in regions with intact basal lamina and relatively normal intercellular spaces, as well as in areas in which the keratinocytes were more severely disrupted. It appeared that the epithelium of lesions which presented clinically as erosive lichen planus contained the largest number of mast cells and other mononuclear cells; however, no quantitation of cell types was performed. In terms of individual cell ultrastructure, the mast cells had nuclei which conformed generally to the shape of the cell, while containing one or more small indentations. Nucleoli were commonly present, as was peripheral clumping of chromatin. The majority of the intraepithelial mast cells had quite well-developed Golgi regions, usually located in an area of nuclear indentation (Figs. 1, 3, 4). These Golgi regions contained mast cell granules which appeared to be in an early stage of granule formation [13, 14, 19,20 ]. Also conspicuous in the cytoplasm were profiles of rough endoplasmic reticulum and clusters of free ribosomes (Figs. 1, 3). Microtubules and microfilaments were present, the former most numerous in the Golgi regions (Figs. 1-4). Mitochondria were located in a perinuclear distribution, and were also concentrated in Golgi regions (Figs. 1-4). Although all the intraepithelial mast cells contained mature mast cell granules, these tended to be rather sparsely distributed in most of the cells as compared to the packing of granules seen in normal mature mast cells [18,21]. The finding of several empty or partially empty granule-like structures at the periphery of the mast cells suggested that degranulation had occurred [21, 22] (Figs. 1, 3). Of special interest was the finding of centrioles in several of the intraepithelial mast cells. These organelles were located in the Golgi region of the

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4 June 1975 cell (Figs. 1, 4). The cells containing the centrioles also had relatively few mature granules sparsely distributed within the cytoplasm, and had a prominent rough endoplasmic reticulum. However, no mitotic figures were seen in mast cells. DISCUSSION The ultrastructural observations reported herein indicate that intraepithelial mast cells are contained within the " lichenoid infiltrate" of the epithelium in gingival lichen planus, along with lymphocytes and macrophages. Although the presence of intraepithelial mast cells in lichen planus has been noted before on both a light [5] and electron [23] microscopic level, these studies did not consider the functional status of the cells. The present ultrastructural study reveals that the intraepithelial mast cells have cytologic features consistent with active synthesis and release of granular components. Moreover, these cells are rather unusual in the fact that they sometimes contain centrioles, since these organelles have previously been described only in mast cells at an early stage of differentiation [15] and have not been mentioned in descriptions of mature mast cells in adult organisms [1-3,18,21]. It has been suggested [23] that an initial infiltration of mononuclear cells into the epithelium in oral lichen planus is responsible for the epithelial changes seen in this disease. It is difficult, however, to understand how such a conclusion can be drawn from a static morphologic study, since it is conceivable, for example, that subtle changes in keratinocytes may be initially responsible for eliciting the mononuclear infiltration. Nevertheless, it seems reasonable to assume that the intraepithelial mononuclear cells have some role in the pathogenesis of the disease. In the case of mast cells, this role is difficult to establish since a unified concept of mast cell function has yet to be established [1]. If one considers the biologically active substances contained within the granules, histamine and heparin would appear to have little effect in an intraepithelial site, whereas the chymotrypsin and trypsin-like proteases [24,25 ] might conceivably have a role in epithelial disruption. This possibility was suggested in a consideration of the role of intraepithelial mast cells in the pathogenesis of human periodontal disease [7] where it was noted that classic methods for obtaining experimental epithelial cell separation have, in fact, utilized trypsin [26 ]. INTRAEPITHELIAL MAST CELLS IN LICHEN PLANUS 439 The finding of centrioles in intraepithelial mast cells with relatively few, sparsely distributed granules, well-developed Golgi regions, and prominent rough endoplasmic reticulum is particularly intriguing. Mast cells with these cytologic features have previously been described only in the early stages of differentiation from precursor " blast" cells [15 ]. Since studies have been reported which indicate that mast cells may be derived from sensitized lymphoid cells subject to antigenic stimulation [27 ], it is tempting to speculate that a certain num her of intraepithelial mast cells in gingival lichen planus may be derived from lymphoid cells already present in the epithelial layer, as a consequence of immunologic interactions. It is clear, however, that evidence to substantiate such a speculation can come only from more dynamic studies of involved tissue. REFERENCES 1. Benditt EP, Lagunoff D: The mast cell: its structure and function. Prog Allergy 8: , Smith DE: The tissue mast cell. lnt Rev Cytol 14: , Selye H: The Mast Cells. Washington, Butterworths, Carranza FA Jr, Cabrini RL: Mast cells in human gingiva. Oral Surg 8: , Hall WB: Mast cells in desquamative gingivitis, lichen planus, and pemphigoid. Oral Surg 28: , Barnett ML: Mast cells in the epithelial layer of human gingiva. J Ultrastruct Res 43: , Barnett ML: The fine structure of human epithelial mast cells in periodontal disease. J Periodont Res 8: , Colvin RB, Dvorak AM, Dvorak HF: Mast cells in the cortical tubular epithelium and interstitium in human renal disease. Hum Pathol 5: , Okun MR, Donnellan B: Ultrastructural relationship of melanocytes to mast cells and "melanophages" in a lesion of alopecia mucinosa. J Invest Dermatol 59: , Miller HRP, Jarrett WFH: Immune reactions in mucous membranes. I. Intestinal mast cell response during helminth expulsion in the rat. Immunology 20: , Murray M, Miller HRP, Jarrett WFH: The globule leukocyte and its derivation from the subepithelial mast cell. Lab Invest 19: , Shklar G, McCarthy P: The oral lesions of lichen planus. Oral Surg 14: , Combs, JW: Maturation of rat mast cells. An electron microscopic study. J Cell Biol 31: , Combs JW: An electron microscopic study of mouse mast cells arising in vivo and in vitro. J Cell Biol 48: , Miller HRP: Immune reactions in mucous membranes. 11. The differentiation of intestinal mast FIG. 2. Intraepithelial mast cell, lichen planus. This mast cell (me), in the lower stratum spinosum, has the close packing of granules more characteristic of a mature mast cell. The epithelium is quite disrupted, with widened intercellular spaces (ics) containing an amorphous electron-dense material. The keratinocytes have large intracytoplasmic spaces (sp) containing a similar material. Desmosomes and tonofilaments are quite sparsely distributed ( x 10,000). FIG. 3. Intraepithelial mast cell, lichen planus. The mast cell (me) is in close association with 3 m nonuclear cells, 2 of which appear to be lymphocytes (cl, c2) and the third of which (c3) cannot be definitively identified. The mast cell has relatively few, sparsely distributed granules, and a prominent Golgi zone. Mitochondria are numerous. Cytofilaments and rough endoplasmic reticulum are conspicuous (x 10,400). FIG. 4. lntraepithelial mast _c~ll, Jichen planus. A higher magnification of the well-developed Golgi region of the cell shown in Fig. 1. Note the centriole (c), mitochondria (m), and mast cell granules (gr) in various stages of development. Microtubules are especially conspicuous adjacent to the nucleus (nu), and microfilaments are distributed more widely. Free and membrane-associated ribosomes can also be seen (x 35,600).

5 440 BARNETT cells during helminth expulsion in the rat. Lab Invest 24: , Ito S, Karnovsky M: Formaldehyde-glutaraldehyde fixative containing trinitro compounds. J Cell Bioi 39:168a, Reynolds ES: The use of lead citrate at high ph as an electron opaque stain in electron microscopy. J Cell Biol17: , Weinstock A, Albright JT: The fine structure of mast cells in normal human gingiva. J Ultrastruct Res 17: , Fujita H, Asagami C, Suetomi Y, Araki I, Kinoshita K: An ultrastructural study of mast granule formation in embryonic skin. Acta Derm Venereol (Stockh)52: Kobayasi T, Asboe-Hansen G: DegranulatiOn and regranulation of human mast cells. An electron microscopic _study of the whealing reaction in urticaria pigmentosa. Acta Derm Venereol (Stockh) 49: , Lagunoff D: Contributions of electron microscopy to Vol. 64, No.6 the study of mast cells. J lnvest Dermatol 58: , Taichman NS: Ultrastructural alterations in guinea pig mast cells during anaphylaxis. Int Arch Allergy Appl Immunol 40: , Hashimoto K, Dibella RJ, Shklar G, Lever WF: Electron microscopic studies of oral lichen planus. G Ital Dermatol107: , Lapmoff D: The properties of mast cell proteases. Biochem PharmacolSuppl17 -:221-~27, 196"8 25. Vensel WH, Komender J, Barnard EA: Non-pancreatic proteases of the chymotrypsin family. II. Two proteases from a mouse mast cell tumor. Biochim Biophys Acta 250: , Moscona A, Moscona H: The dissociation and aggregation of cells from organ rudiments of the early chick embryo. J Anat 86: , Ginsburg H, Lagunoff D: The in vitro differentiation of mast cells. Cultures of cells from immunized mouse lymph nodes and thoracic duct lymph on fibroblast monolayers. J Cell Bioi 35: , 1967