International Journal of Surgical Pathology

International Journal of Surgical Pathology http://ijs.sagepub.com

Merkel Cells in the Oral Mucosa Alberto Righi, Christine M. Betts, Claudio Marchetti, Gianluca Marucci, Lucio Montebugnoli, Carlo Prati, Leonardo H. U. Eusebi, Luciana Muzzi, Teresa Ragazzini and Maria P. Foschini Int J Surg Pathol 2006; 14; 206 DOI: 10.1177/1066896906290053 The online version of this article can be found at: http://ijs.sagepub.com/cgi/content/abstract/14/3/206

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Merkel Cells in the Oral Mucosa

International Journal of Surgical Pathology Volume 14 Number 3 July 2006 206-211 © 2006 Sage Publications 10.1177/1066896906290053 http://ijsp.sagepub.com hosted at http://online.sagepub.com

Alberto Righi, MD, Christine M. Betts, MD, Claudio Marchetti, MD, Gianluca Marucci, MD, Lucio Montebugnoli, MD, Carlo Prati, MD, Leonardo H. U. Eusebi, MD, Luciana Muzzi, BSc, Teresa Ragazzini, BSc, and Maria P. Foschini, MD Ninety-eight consecutive surgical biopsies of oral mucosa from 96 patients were evaluated immunohistochemically with an anti–cytokeratin 20 (CK 20) antibody to evidence Merkel cells (MC). Fifteen cases, showing the highest number of MC, were additionally studied with chromogranin A, S-100 protein, neuro filaments, epithelial membrane antigen, and double immunostaining for CK 20 and Ki67 antibodies to evaluate MC proliferation. Electron microscopy was performed in 2 cases. MC were observed in 58 cases. The highest number of MC was found in the gingival, buccal,

and palate mucosa, especially in chronically damaged oral mucosa (lichen and chronic aspecific inflammation) as well as in the mucosa overlying tumors rather than in normal or acute inflammation. MC were not observed in dysplastic or neoplastic epithelium. MC showed evidence of proliferation, as demonstrated by Ki67 positivity, in 3 cases. In conclusion, MC appear to play a role in the reparative processes of oral mucosa.

M

Distribution of MC in normal and pathological conditions has been extensively studied in adult and prenatal human skin.5-8 On the contrary, only a few studies have been published9-11 on MC of the human oral mucosa. The aim of the present article was to study the presence and distribution of MC in normal and pathological oral mucosa.

erkel cells (MC) are endocrine cells originally identified by Merkel in 1875, who described “helle Zellen” (clear cells) associated with nerve fibers1 in the basal layers of human and animal epidermis as well as in oral mucosa. Merkel regarded the newly identified cells as having sensory touch function, and he named these same cells “Tastzellen” (touch cells). At electron microscopy, MC are characterized by small, electron-dense endocrine-like granules, intermediate filaments, and occasional desmosomes.2,3 The endocrine nature of MC is further proven by their immunoreactivity with various endocrine markers, especially chromogranin A and synaptophysin, which are the most frequently found. In addition, MC express numerous keratins (CK), including CK 7, 8, 18, 19, and 20. The latter (CK 20) is of particular value, since it is exclusive to MC in normal squamous epithelium.4 From the Section of Anatomic Pathology, Bellaria Hospital, University of Bologna, Bologna, Italy (AR, GM, LHUE, LM, TR, MPF), the Department of Experimental Pathology, University of Bologna, Bologna, Italy (CMB), and the Department of Dental Sciences, University of Bologna, Bologna, Italy (CM, LM, CP). Address correspondence to: Maria P. Foschini, MD, Anatomia Patologica, Ospedale Bellaria, Via Altura, 3, 40139 Bologna, Italy; e-mail: [email protected].

Keywords: oral mucosa; Merkel cell; proliferation; reparative process

Materials and Methods Ninety-eight consecutive biopsies of the oral cavity from 96 patients were obtained from the files of the Section of Pathology of the University of Bologna at Bellaria Hospital. The age of the patients ranged from 3 to 84 years (mean, 50 years). Biopsies were obtained from different sites of the oral mucosa: 46 mandibular gingiva, 19 palate (15 hard palate and 4 soft palate), 13 tongue, 12 cheek, and 8 buccal mucosa. All tissues were routinely fixed in buffered formalin, embedded in paraffin, and stained with hematoxylin and eosin (H&E). From each paraffin block, sections were also immunohistochemically stained for CK 20 (clone KS 20.8, dilution 1:40; Dako, Carpinteria, Calif). Processing was performed in an automatic stainer (Autostainer; Dako) using

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Merkel Cells in the Oral Mucosa / Righi et al

Table 1.

Merkel Cells at 6 Anatomical Sites in Oral Mucosa

Gingiva No. (%) Range Mean, no. of MC per case Location Shape

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Hard or Soft Palate

29/46 (63) 1-80 13 B 25/29 S 22/29 O 26/29 D 23/29

Buccal Mucosa

Tongue

Cheek

5/8 (62) 2-19 9 B 5/5 S 2/5 O 5/5 D 3/5

4/13 (31) 1-9 5 B 4/4 S 0/4 O 4/4 D 0/4

4/12 (33) 1 1 B 4/4 S 0/4 O 4/4 D 0/4

16/19 (84) 1-40 14 B 16/16 S 4/16 O 14/16 D 12/16

Note: MC, Merkel cells; B, basal; S, suprabasal; O, oval cells; D, dendritic cells.

Table 2. Normal No. (%) 7/14 (50) Range 1-27 Mean, no. of MC per case 8 Location B 7/7 S 5/7 Shape O 7/7 D 5/7

Merkel Cells in Normal and Pathological Oral Mucosa Acute Inflammation

Chronic Aspecific Inflammation

Lichen

8/16 (50) 3-32 11 B 8/8 S 7/8 O 7/8 D 7/8

22/28 (79) 1-80 16 B 22/22 S 14/22 O 20/22 D 14/22

5/7 (71) 1-14 6 B 5/5 S 2/5 O 5/5 D 2/5

Epithelium Overlying Tumors 12/15 (80) 1-40 13 B 12/12 S 4/12 O 11/12 D 7/12

In Situ Dysplasia Carcinoma 0/5 — — — — — —

5/13 (38)a 1-16 8 B 5/5 S 0/5 O 3/5 D 5/5

Note: MC, Merkel cells; B, basal; S, suprabasal; O, oval cells; D, dendritic cells. a. MC present in the mucosa adjacent to the lesion only.

biotinylated coat antipolyvalent and streptavidin peroxidase (LabVision Corp, Fremont, Calif). Dako Liquid DAB Substrate-Chromogen system was used as the reveal system. In all cases, the number of CK 20–positive cells per 300 basal keratinocytes was counted. In addition, specific morphological features of CK 20 cells (oval vs dendritic), localization (basal vs suprabasal), and distribution (single cells vs nests of cells) were evaluated. The localization was considered basal when CK 20 cells were located within the first 2 layers above basal keratinocytes and suprabasal when they were located in the more superficial layers. Fifteen selected cases, showing the highest number of CK 20–positive cells, were also stained for epithelial membrane antigen (EMA) (clone E29, dilution 1:600; Dako), chromogranin A (clone LK2H10, dilution 1:200; Biogenex, San Ramon, Calif) S-100 protein (polyclonal Z311, dilution 1:1500; Dako) and neurofilaments (clone NE14, dilution 1:6; Biogenex). In the same 15 cases, double staining with anti–CK 20 antibody and anti–Ki67 antibody (clone MIB 1, diluted 1:200; Dako) was performed as follows:

streptavidin was conjugated with alkaline phosphatase and 2 different chromogens: fast red and Dako Liquid DAB Substrate-Chromogen system. For electron microscopy, small blocks were microdissected from paraffin-embedded samples in 2 cases where CK 20–positive cells were numerous. These small blocks were dewaxed by overnight immersion in xylol and rehydrated to phosphate buffer, through a graded series of alcohol dilutions. Biopsies were then refixed in buffered glutaraldehyde, postfixed in OsO4, dehydrated in alcohol, and embedded in Epon 812 (Fort Washington, Pa). Thick sections stained with toluidine blue were used to select areas to examine. Thin sections were stained with uranyl acetate and Reynold’s lead citrate, and observed in a Philips CM 10 TEM (Eindhoven, the Netherlands).

Results Histology. On H&E-stained sections, cells showing clear cytoplasm were occasionally seen after a careful search of the basal layer.


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Immunohistochemistry. CK 20–positive cells (MC) were detected in 58 biopsies. MC location. MC were basally located in 56 biopsies. In only 2 biopsies were they located in more superficial layers, and in no cases were they found in the chorion. MC were generally observed individually located (43 biopsies) within basal keratinocytes. In 15 biopsies, MC formed nests located at the tip of the rete ridges. The shape of MC ranged from oval (in 53 biopsies) to dendritic (in 38 biopsies). MC number according to site. MC were most frequently present in the hard and soft palate (mean, 14 MC per case), followed by the mandibular gingiva (mean, 13 MC per case) and buccal mucosa (mean, 9 MC per case) (Table 1). MC presence according to anatomoclinical findings. In normal oral mucosa, MC were observed in 50% of biopsies, distributed as single cells along the basal layer of the epithelium, and averaging 8 in number. In acute inflammation, MC were present in 50% of biopsies as occasional single cells (Table 2; Fig. 1). In chronically damaged mucosa (lichen and chronic aspecific inflammation), MC were more frequently detected: they were observed in 71% of cases of lichen planus, in 79% of chronic aspecific inflammation, and in 80% of mucosa overlying tumors. In these latter cases, MC were confined to the squamous epithelium, and in no cases were MC found within the neoplastic tissue of the underlying tumor. In all these cases, MC were distributed as single elements or formed nests located at the tips of the rete ridges and averaging 15 in number. In no cases were MC observed within the dysplastic or neoplastic oral mucosa. Rare MC were found in the “normal” epithelium surrounding 5 of 13 in situ carcinomas (38% of cases). Additional immunohistochemistry. In the 15 cases with additional markers, the same cells that stained for CK 20 showed strong expression for chromogranin A (Fig. 2a) and EMA (Fig. 2b), while they were negative for neurofilaments or S-100 protein.

Figure 1. CK 20–positive Merkel cells are rare in acute inflammation and located at the margins of the lesion (A), and they are numerous in chronic inflammation, distributed either in a continuous layer (B) or in nests (C).

Double immunostaining. Double immunostaining showed that MC are intermingled with basal keratinocytes (Fig. 3a). Occasional MC (no more than 3 in 3 different cases among all 15 cases) showing cytoplasmic positivity for CK 20 also evidenced nuclear positivity for Ki67, similarly to basal keratinocytes (Fig. 3b).



Figure 2. Merkel cells positive for chromogranin A (A) and epithelial membrane antigen (B).

Ultrastructural features. In both cases studied, cells showing intracytoplasmic dense core granules, and finely dispersed paranuclear intermediate filaments were observed (Fig. 4). Granules ranged from 80 to 100 nm in diameter. Filaments were sparse, resulting in a clear cytoplasm. No direct relationship with nerve endings was envisaged.

Discussion The histological, immunohistochemical, and ultrastructural features of the CK 20–positive cells were consistent with those of Merkel cells of the skin.2

209

Figure 3. (A) Double immunostaining highlights Merkel cells in close contact with basal keratinocytes, the latter positive for Ki67 (CK 20 in red; Ki67 in brown); (B) double immunostaining demonstrates Merkel cells with nuclei stained for Ki67 (CK 20 in red; Ki67 in brown).

The present study shows that MC are normally present in oral epithelium. The mean number of 8 MC every 300 basal keratinocytes was far higher than that observed in the skin.5 MC were more numerous in palatal, gingival, and buccal mucosa, the areas that more frequently undergo masticatory trauma.9,11 Further demonstration of the importance of masticatory load was provided by Kingsmill et al,10 who found MC increase in oral mucosa of edentulous subjects. MC showed a different distribution in acutely damaged epithelium and in chronic lesions. MC were absent in areas of acute inflammation. Similarly, MC


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Figure 4. Ultrastructurally, Merkel cells contain small, endocrine-like cytoplasmic granules and intermediate filaments.

were absent in squamous epithelium with dysplasia or in situ carcinoma. In no case were they found in tumors, in contrast to what was previously demonstrated in a case of peripheral ameloblastoma.12 On the contrary, in chronically damaged mucosa, MC were numerous and frequently nested at the tip of the rete ridges. The present findings, therefore, indicate that MC number increases in chronically inflamed oral mucosa, thus suggesting a role of MC in reparative processes of the oral epithelium. In keeping with this view, we found MC with positive Ki67 (a marker of cell proliferation13,14) in 3 cases. Presence of rare proliferating MC has been described by Merot et al15 and by Vaigot et al16 in rabbit and murine skin. Moll et al17 failed to demonstrate Ki67-positive MC in normal human fetal and adult skins, suggesting that MC are postmitotic cells derived from undifferentiated keratinocytes. Admittedly, proliferating MC in the present cases were rare and were found in chronically damaged oral mucosa, where the number of MC was increased with respect to the normal mucosa, although it appeared that MC could proliferate within the squamous epithelium in the same fashion as basal keratinocytes. MC function has not yet been completely elucidated. In human skin, MC act as mechanoreceptors, transforming mechanical stimuli to action potential in the afferent nerve fibers.18 A second possible function is the endocrine-paracrine function involved in the

cell proliferation of the related epithelium.19,20 Tachibana et al21,22 demonstrated exocytosis in rodent palatine mucosa. In vitro, neuropeptides produced by MC (vasoactive intestinal polypeptide and substance P) are known to be able to promote cell growth and differentiation of various cutaneous cells such as keratinocytes, fibroblasts, and endothelial cells.23,24 In the present series, MC were located in close contact with proliferating basal keratinocytes and were themselves positive for Ki67, indicating that even in human oral mucosa, MC may play a role in epithelial cell proliferation. In addition, MC of the present cases were more numerous in cases showing reparative/proliferative epithelium. This parallels with what is seen in the skin, where MC are numerous in chronically damaged skin such as in actinic dermatoses, chronic UV exposure,7,25 and prurigo nodularis,8 when reparative epidermal processes are very active. On the contrary, MC disappear in acute inflammatory lesions, leading Moll et al7 to suggest that MC are destroyed in acutely damaged skin. In conclusion, it appears that MC are numerous in the oral cavity and are mainly located in areas that usually undergo masticatory load, such as the gingival and buccal mucosa and palate. In addition, MC number increases in chronically damaged mucosa, thus suggesting a possible endocrine-paracrine trophic function in reparative and regeneration processes.

Acknowledgments This work was financed with grants from the University of Bologna ex 60% (to MPF).

References 1. Merkel F. Tastzellen und Tastkorperchen bei den Haustieren und beim Menschen. Arch Microsc Anat. 1875;11:636-652. 2. Frigerio B, Capella C, Eusebi V, Tenti P, Azzopardi JG. Merkel cell carcinoma of the skin: the structure and origin of normal Merkel cells. Histopathology. 1983; 7(2):229-249. 3. Winkelmann RK, Breathnach AS. The Merkel cell. J Invest Dermatol. 1973;60:2-15. 4. Moll I, Kuhn C, Moll R. Cytokeratin 20 is a general marker of cutaneous Merkel cells while certain neuronal proteins are absent. J Invest Dermatol. 1995; 104:910-915. 5. Collina G, Eusebi V, Capella C, Rosai J. Merkel cell differentiation in trichoblastoma. Virchows Arch. 1998; 433(4):291-296.



6. Foschini MP, Eusebi V. Divergent differentiation in endocrine and nonendocrine tumors of the skin. Semin Diagn Pathol. 2000;17(2):162-168. 7. Moll I, Roessler M, Brandner JM, Eispert AC, Houdek P, Moll R. Human Merkel cells—aspects of cell biology, distribution and functions. Eur J Cell Biol.2005;84: 259-271. 8. Nahass GT, Penneys NS. Merkel cells and prurigo nodularis. J Am Acad Dermatol. 1994;31:86-88. 9. Barrett AW, Cort EM, Patel P, Berkovitz BKB. An immunohistochemical study of cytokeratin 20 in human and mammalian oral epithelium. Arch Oral Biol. 2000; 45(10):879-887. 10. Kingsmill VJ, Berkovitz BKB, Barrett AW. An immunohistochemical analysis of human Merkel cell density in gingival epithelium from dentate and edentulous subjects. Arch Oral Biol. 2005;50:883-887. 11. Tachibana T, Kamegai T, Takahashi N, Nawa T. Evidence of polymorphism of Merkel cells in the adult human oral mucosa. Arch Hist Cytol. 1998;61:115-124. 12. Marucci G, Betts CM, Michal M, Foschini MP. Peripheral ameloblastoma with Merkel cells. Virchows Arch. 2005; 446(2):204-205. 13. Brown DC, Gatter KC. Monoclonal antibody Ki67. Its use in histopathology. Histopathology. 1990;17: 489-507. 14. Gerdes J, Li L, Schlueter C, Duchrow M, et al. Immunobiochemical and molecular biologic characterization of the cell proliferation-associated nuclear antigen that is defined by monoclonal antibody Ki-67. Am J Pathol. 1991;138: 867-873. 15. Merot Y, Carraux P, Saurat JH. Merkel cell mitoses in vibrissae: an ultrastructural study. J Anat. 987;153: 241-244.

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16. Vaigot P, Pisani A, Darmon YM, Ortonne JP. The majority of epidermal Merkel cells are non proliferative: a quantitative immunofluorescence analysis. Acta Derm Venereol (Stockh). 1987;67:515-541. 17. Moll I, Zieger W, Schmets M. Proliferative Merkel cells were not detected in human skin. Arch Dermatol Res. 1996; 288:184-187. 18. Chen SY, Gerson S, Meyer J. The fusion of Merkel cells granules with a synapse-like structure. J Invest Dermatol. 1973;61:290-292. 19. Halata Z, Grim M, Bauman KI. Friedrich Sigmund Merkel and his “Merkel cell,” morphology, development and physiology: review and new results. Anat Rec A Discov Mol Cell Evol Biol. 2003;271(1):225-239. 20. Nunzi MG, Pisarek A, Mugnaini E. Merkel cells, corpuscular nerve endings and free nerve endings in the mouse palatine mucosa express three subtypes of vesicular glutamate transporters. J Neurocyt. 2004;33:359-376. 21. Tachibana T, Yamamoto H, Takahashi N, et al. Polymorphism of Merkel cells in the rodent palatine mucosa: immunohistochemical and ultrastructural studies. Arch Histol Cytol. 1997; 60:379-389. 22. Tachibana T, Nawa T. Recent progress in studies on Merkel cell biology. Anat Sci Int. 2002;77:26-33. 23. Haegerstrand A, Jonzon B, Dalsgaard CJ, Nilsson J. Vasoactive intestinal polypeptide stimulates cell proliferation and adenylate cyclase activity of cultured human keratinocytes. Proc Natl Acad Sci USA. 1989;86:5993-5996. 24. Nilsson J, Euler AN, Dalsgaard CJ. Stimulation of connective tissue cell growth by substance P and substance K. Nature. 1985;315:61-63. 25. Moll I, Bladt U, Jung EG. Presence of Merkel cells in sun-exposed and not sun-exposed skin: a quantitative study. Arch Dermatol Res. 1990;282:213-216.


International Journal of Surgical Pathology

International Journal of Surgical Pathology

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