Architectural Organization of Filiform Papillae in Normal

OBSERVATION Architectural Organization of Filiform Papillae in Normal and Black Hairy Tongue Epithelium Dissection of Differentiation Pathways in a Complex Human Epithelium According to Their Patterns of Keratin Expression

Motomu Manabe, MD, PhD; Henry W. Lim, MD; Martin Winzer, MD; Cynthia A. Loomis, MD, PhD

Background: An inadequate understanding of the lying the primary papillae and between the individual pri- complex morphologic characteristics of human fili- mary papillae express esophageal-type keratins. In black form papillae has hampered the histopathological hairy tongue disease, there is a marked retention of sec- characterization of disorders affecting tongue kera- ondary papillary cells expressing hair-type keratins. tinization. To better define the 3-dimensional cyto- architecture of tongue epithelium, we performed Conclusions: Using a panel of antikeratin probes, we detailed immunohistochemical analyses of normal and define the precise topographical localization of cell black hairy tongue tissues using a panel of antikeratin populations undergoing 3 distinct differentiation pro- antibodies. grams in dorsal tongue epithelium. Comparative analyses of black hairy tongue specimens indicate that defec- Observations: The dome-shaped base of the human fi- tive desquamation of the cells in the central column of liform papilla (primary papilla) is surmounted by 3 to 8 filiform papillae results in the formation of highly elon- elongated structures (secondary papillae). These second- gated, cornified spines or “hairs”—the hallmark of this ary papillae are composed of a central column of epithe- disease. lial cells expressing hair-type keratins and an outer rim of cells expressing skin-type keratins. The epithelium over- Arch Dermatol. 1999;135:177-181

HE DORSAL surface of mam- cumulative data indicate that, in addition malian tongue is covered by to the ubiquitous expression of the strati- densely packed filiform pa- fied epithelial-type keratins (K5 and K14), pillae. The shape and size different regions of the tongue epithe- of these papillae vary mark- lium display distinct patterns of keratin ex- edlyT from species to species.1-8 In hu- pression. The nonkeratinized epithelium mans, the papillary architecture is more of the lateral and ventral surfaces of the complex, consisting of a central body sur- tongue, as well as the epithelium cover- rounded by several threadlike cornified ing the lateral aspects of filiform and projections, often referred to as second- fungiform papillae and the interpapillary ary papillae.5,9 Morphologically, human mucosa of the dorsal tongue, express tongue epithelium seems to be divided esophageal-type keratins (K4 and K13). In into discrete domains that undergo dis- contrast, the orthokeratinized epithe- tinct pathways of terminal differentia- lium overlying the tips of filiform papil- tion, similar to what occurs in rodent and lae makes skin-type keratins (K1 and K10), From the Departments of cow tongue epithelium. However, the mor- and the epithelium covering taste buds Dermatology, Akita University phologic compartments of the human synthesizes simple epithelial-type kerat- School of Medicine, Akita, tongue have not been as well character- ins (K8, K18, and K19). Finally, we and Japan (Dr Manabe), and Henry ized as those of the rodent because of their others14-16 have shown that tongue epithe- Ford Hospital, Detroit, Mich greater complexity. lium also produces an acidic hard-type (Dr Lim); the Keratins are a heterogeneous family keratin, characteristic of hair- or nail- Dermatohistopathology of polypeptides that form a subclass of in- type differentiation. Laboratory, Westerstede, Germany (Dr Winzer); and The termediate filaments. They serve as excel- In this article, we extend our immu- Ronald Perelman Department of lent markers for various pathways of epi- nolocalization studies on the structure and Dermatology, New York thelial differentiation and have been used compartmentalization of the human fili- University School of Medicine, to define discrete populations of keratino- form papilla and begin to explore the role NY (Dr Loomis). cytes within the tongue epithelium.10-13 The of such compartments in human disease.

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MATERIALS AND METHODS SPECIFICITY OF THE AE 20 ANTIBODY FOR HUMAN EPIDERMAL KERATIN HUMAN TISSUE In our prior study of human tongue epithelium,7 in- Normal human tongues were obtained from au- direct immunofluorescent staining was performed us- topsy within 48 hours of death. The 3 biopsy speci- ing the monoclonal antibody AE 2, which recognizes mens of BHT were obtained from former patients markers of skin-type differentiation, K1 and K10.19 Un- of Manhattan Veterans Affairs Medical Center fortunately, AE 2 cross-reacts with filaggrin, a major com- in New York as well as Medical University of Lu¨ beck ponent of keratohyalin granules (data not shown), mak- in Germany. ing it difficult to localize the skin-type keratins. To obtain more definitive immunolocalization data, we generated ANTIKERATIN ANTIBODIES a monoclonal antibody, AE 20, that reacts specifically with Detailed characterizations of the monoclonal anti- the basic 67-kd human epidermal keratin, K1, as as- body AE 8, which reacts specifically with the sessed by immunoblotting (data not shown). No AE 20 esophageal-type keratin K13, and the antibody AE immunoreactivity was detected in keratin extracts from 13, which reacts with 44- to 46-kd acidic hair ker- a variety of other tissues and cultured cells tested (data atins, are described elsewhere.17,18 The AE 20 anti- not shown). body was generated against trypsinized human skin cells according to the methods described pre- SEVERAL DISTINCT DIFFERENTIATION 19 viously. The antibody to K6 was generously pro- DOMAINS OF CROWN-SHAPED vided by Dennis Roop, PhD, Baylor University, HUMAN FILIFORM PAPILLA Houston, Tex.

KERATIN EXTRACTION, The human filiform papilla consists of an elevated con- GEL ELECTROPHORESIS, nective tissue core covered by a partially keratinized strati- AND IMMUNOBLOTTING fied epithelium. Small connective tissue protrusions ema- nate from the top of this central core and are surmounted Normal human epidermis and cultured epithelial by a column of epithelial cells undergoing cornification. cells from various tissues were first extracted with These elongated cornified spines are referred to as sec- 25-mmol/L Tris-hydrochloride, 0.6-mol/L po- ondary papillae and characteristically tilt toward the phar- tassium chloride, 1% Triton X-100 supplemented 20 ynx. In contrast to the papillary epithelium, the inter- with 5 protease inhibitors. The residual aqueous- papillary surface is nonkeratinized. insoluble cytoskeletal preparation, containing To facilitate the topological analysis of this com- mainly keratin proteins, was then solubilized with 2% sodium dodecyl sulfate in 25-mmol/L plex structure, we used a panel of monoclonal antibod- Tris-hydrochloride (pH 7.4) as described.20 ies recognizing keratins that are characteristic of differ- One-dimensional sodium dodecyl sulfate– ent types of stratified epithelia. The AE 8 monoclonal polyacrylamide gel electrophoresis and subsequent antibody specifically reacts with the K4 keratin, which immunoblotting were performed according to the is expressed in nonkeratinized, stratified epithelia such methods described previously.21 as in the esophagus. The AE 13 antibody recognizes acidic hair keratins, which are expressed in hair cortex, nail IMMUNOHISTOCHEMICAL STAINING plate, and mouse tongue filiform papillae.14,17 The AE 20 antibody, described above, recognizes K1, which is char- The biopsy specimens were embedded in OCT com- pound (Ty Miles Inc, Westchester, Ill), snap frozen acteristic of keratinized epithelia such as skin. The an- in liquid nitrogen, and cut into 6-µm cryostat sec- tibody to K6 recognizes a keratin that is normally ex- tions. Sections were stained by the indirect immu- pressed at a few restricted sites of the body, including nofluorescent and immunoperoxidase technique.19,22 volar skin, foreskin, oral mucosa, and outer root sheath cells of the hair follicle, as well as by all stratified epithelial cells undergoing hyperproliferation.23-25 Using this panel of antikeratin antibodies, we con- firmed that the epithelium covering filiform papillae is We present the characterization of a new antikeratin subdivided into at least 3 distinct cell domains (data not K1 monoclonal antibody and its use to definitively shown). As reported previously, the antiesophageal kera- localize the skin-type compartment in human tongue tin antibody, AE 8, reacted with the nonkeratinizing epi- epithelium. In addition, we provide a more detailed thelia between the filiform papillae as well as between description of the 3-dimensional organization of the the cornified hairlike projections. The AE 20 immuno- separate domains within human filiform papillae. reactivity was confined to cells at the bases of the corni- Finally, we demonstrate that the hairlike projections fied spines, indicating that the skin-type keratin K1 is re- observed in black hairy tongue disease (BHT) are due stricted to this cell population. The AE 13 antihair keratin primarily to the formation of abnormally long exten- antibody reacted only with those cells directly beneath sions of the “hair compartment” of the filiform the cornified spines, adjacent to the AE 20–positive cells. papillae. Immunostaining with the antibody to K6 was uni-

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P2 P2 B

IP A C

e IP h IP s

Normal Tongue Black Hairy Tongue

Figure 1. A schematic summary of filiform papillae in normal and black hairy A B tongue epithelium. Horizontal (A) and longitudinal (B) sections of normal dorsal tongue epithelium, and a longitudinal section (C) of black hairy tongue. Areas expressing hair- (h), skin- (s), and esophageal- (e) type keratins are the hatched, filled, and dotted areas, respectively.

formly positive in the suprabasal cells of the dorsal tongue, similar to other oral epithelium.13 Detailed analysis of horizontal sections of human filiform papillae allowed us to further refine our understanding of the 3-dimensional architecture of the filiform papillae and the topological locales of different epithelial domains (data not shown). Within the large central area surrounded by a ring of 5 to 12 cornified spines are AE 8–positive cells, which synthesize esophageal-type keratins. The interpapillary epithelial cells likewise pro- C D duce esophageal-type keratins, but they appear to con- stitute an esophageal-type compartment distinct from the Figure 2. Immunofluorescent staining of black hairy tongue epithelium. Vertical sections are stained with AE 8 (A), AE 20 (B), or AE 13 (C). Panel D AE 8–positive papillary epithelium. A thin band of AE is a phase contrast image of panel C. P1 indicates primary filiform papillae; 20–positive cells that undergo skin-type differentiation P2, secondary filiform papillae; and IP, interpapillary epithelium. are distributed at the base of each of the cornified spines, encircling the cluster of AE 13–positive cells. Clusters of AE 13–positive cells, as well as their overlying corni- are AE 8–positive, indicating the presence of esophageal- fied spines, are distributed in a ring at the periphery of type keratins (Figure 2, A). The column of cells di- the filiform papillae. These cells synthesize a “hair” kera- rectly beneath the cornified spines are AE 20–positive, tin and undergo hair-type differentiation. Thus, the hu- reflecting the presence of skin-type keratins (Figure 2, man filiform papilla is essentially crown shaped, with the B). The cells adjacent to the AE 20–positive cells are outer rim being formed by the ring of hairlike spines. A stained with AE 13 and thus undergo hair- and nail- scheme of the histological architecture of human fili- type differentiation (Figure 2, C and D). Our studies could form papillae is shown in Figure 1. not exclude, however, the possibility that the entire filiform papilla is involved, resulting in mild hyperkera- ABERRANT ELONGATION OF THE HAIRLIKE tosis in both the skin-type and central intraspinous esoph- DOMAIN IN BHT TONGUE EPITHELIUM ageal-type domains. Interestingly, the interpapillary esophageal domain (the dorsal tongue epithelium be- Since the histological architecture of normal filiform pa- tween the individual primary filiform papillae) shows no pillae has never been described precisely, many com- alterations in its differentiation, based on both morpho- mon denominators make it difficult to differentiate be- logical and immunolocalization criteria. tween normal and BHT epithelium. Furthermore, the most striking finding on routine histological examination of COMMENT BHT is the presence of numerous small fragments of cornified cells, which results from tangential sectioning. To The tongue is covered by a complex epithelium com- avoid this problem, we made an effort to prepare well ori- posed of several functionally distinct cell populations. Our ented longitudinal sections of the BHT biopsy speci- data have enabled us to definitively localize at least 3 mens. Immunofluorescent staining using our panel of unique domains within the tongue epithelium: (1) cells monoclonal antibodies demonstrated that the overall pat- in the secondary filiform papillae that express acidic hair- tern of keratin expression in BHT is similar to that of nor- type keratins, (2) a ring of cells surrounding this hair com- mal tongue epithelium. The cells in the center of the fi- partment that express skin-type keratins, and (3) cells liform papillae as well as those in the interpapillary region overlying the central mound of the primary papillae as

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©1999 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/29/2021 well as between the papillae that express esophageal- descriptions also apply to normal histological character- type keratins. It is fascinating that filiform papillae ex- istics of the tongue. For example, they noted that patho- press both soft (epithelial) keratins and hard (tricho- logical changes associated with BHT include a markedly cytic) keratins. It has been proposed that the coexistence digitated surface, pointed rete ridges, focal parakerato- of these different programs of keratin expression re- sis, focal preservation of the granular zone, ballooned cells flects the dual requirements of tongue epithelium to be with pale cytoplasm in the spinous zone, and the pres- both rigid and flexible to resist friction and expansion ence of both keratohyalin- and trichohyalin-like gran- accompanying tongue movements during food han- ules. However, since normal tongue epithelium consists dling and grooming.14 of distinct but closely juxtaposed differentiation do- While the importance of the above observation re- mains, the above “pathological” findings are also ob- mains to be determined, the existence of 3 distinct do- served in normal tongue. Without a clear understand- mains in filiform papillae raises an interesting question ing of the precise histological topography of the tongue with respect to their cell origin. Do cells in the hair-, skin-, and its specialized epithelial domains, one cannot mean- and esophageal-type domains originate from a common ingfully analyze the pathological processes in tongue dis- pluripotent stem cell, or does each arise from unique eases. Our present study shows that the designation of stem cells located at the base of its domain? Recent re- black hairy tongue is by chance consistent with the spe- sults26-29 suggest that epithelial stem cells, as defined by cific keratin expression in cells that cause the character- their kinetic properties, reside in specific sites within each istic morphologic features. Cells underneath the strik- epithelial tissue. Since stem cells are normally slow cy- ingly elongated cornified spines are indeed derived cling, they can be identified as label-retaining cells after from the hair domain of filiform papillae. However, it re- the labeling of all cycling cells by a continuous admin- mains unclear why the hair-type compartment shows a istration of isotope for a prolonged period. Using this ap- prominent retention of cornified spines, and how the proach, the stem cells of epidermis, intestinal epithe- pathogenic factors implicated in promoting BHT, such lium, corneal epithelium, and hair follicles were shown as smoking, oxidizing agents, and antibiotics, inhibit hair to be located at the bottom of deep rete ridges, the crypts, compartment desquamation. Further studies are neces- the limbal region, and the bulge area, respectively.26,28-30 sary to elucidate the relevant mechanisms. In this re- Interestingly, these findings were further supported by gard, it might be interesting to investigate whether the studies of graft-vs-host disease, in which the sites of medicines that influence hair growth may also cause mor- involvement were thought to correlate precisely in the phologic alterations of the filiform papillae. localization of the stem cells. In graft-vs-host disease, it was shown that cytotoxic lymphocytes infiltrate prefer- Accepted for publication June 22, 1998. entially around the bottom of the epidermal rete ridges, Research for this article has been supported in part the follicular bulge cells, and the anterior and posterior by a Dermatology Foundation Career Development Award shoulders of filiform papillae.31-33 In this regard, our find- (Dr Loomis). ing that filiform papillae can be divided into several dif- We thank Tung-Tien Sun, PhD (New York Univer- ferentiation compartments is noteworthy. It may be im- sity, New York, NY), and Helmut H. Wolff, MD (Medical portant to distinguish the stem cells of these domains University of Lu¨beck in Germany), for their useful discus- because different stem cells may give rise to different neo- sions. plasms of the tongue epithelium. Reprints: Motomu Manabe, MD, PhD, Department of The distinctive spatial arrangement of the hair do- Dermatology, Juntendo University School of Medicine, mains of filiform papillae is of particular interest, given Hondo 1-1-1, Akita 113-8421, Japan (e-mail: manabe that hairs in skin also develop with a patterned distribu- @doc.med.akita-u.ac.jp). tion rather than at random.34-37 For example, a common grouping of follicles is the “trio” in which the primary REFERENCES hair follicle or tylotrich first erupts from the germ, and

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