Eur Arch Otorhinolaryngol (1992) 249 : 380-384
European Archives of
Oto-RhinoLaryngology © Springer-Verlag 1992
Langerhans cells in human middle ear cholesteatomas W.-Y. Chao ~, Y.-T. Jin 2, and C.-C. Huang 3 Departments of 10tolaryngology and 2Pathology, National Cheng Kung University College of Medicine, Tainan, 70101 Taiwan 3Department of Otolaryngology, Columbia University College of Physicians and Surgeons, NewYork, NY 10032, USA Received January 21, 1992 / Accepted June 16, 1992
Summary. Langerhans cells have been found in cholesteatomas for many years. It is believed that they are immunocompetent cells and have the same role in cellmediated immunologic mechanisms in cholesteatoma as well as in skin. This study used the transmission electron microscope to observe the cellular characteristics of Langerhans cells and the apposition p h e n o m e n o n of Langerhans cells with lymphocyte-like cells in human middle ear cholesteatomatous tissue. These findings are evidence for cell-mediated immune responses in middle ear cholesteatomas. In vitro Langerhans cells conditioned medium prepared from Lewis rat skin was used to show its effects on protein synthesis and the differentiation of basal cells. Since the cellular behaviour of basal cells is important in the development and pathogenesis of cholesteatoma, the present study shows that Langerhans cells may have some role in the clinical formation of a cholesteatoma. Since cells extracted from rat skin may have a different response from that of cells from human middle ear cholesteatoma, further investigations are necessary to compare the biological effects of both tissues. Key words: Cholesteatoma - Langerhans cells - Basal cells - T-lymphocytes
Introduction Cholesteatoma is a c o m m o n middle ear disease which is pathologically benign but clinically destructive in the temporal bone. It is composed of epithelial matrix and connective tissue, including granulation tissue and many inflammatory cells. The roles of cellular components in the pathogenesis of cholesteatoma have been studied by many institutes. A m o n g them, Langerhans cells (LC) have been noted for many years, but their relationship with T-lymphocytes has been assumed to be clinically important in the disease process only recently. In the present study transmission electron microscopy was used to study human middle ear cholesteatomas obtained from ear surgery to provide some explanation for the pathogenesis of this disease. Previous studies have shown that LC density in seborrheic keratosis is significantly higher than that in basal cell and squamous cell carcino-
Correspondence to: W.-Y. Chao
mas [4]. To determine whether LC plays a role in promoting differentiation of the epithelial cells of a cholesteatoma, this study was designed to demonstrate that the in vitro cellular behavior of basal cells is influenced by the factors released from LC into the tissue medium. To date, the treatment of a temporal bone cholesteatoma has been surgical removal. Understanding the roles of various cells in cholesteatomas may provide some insight for future management.
Materials and methods Sixteen human middle ear cholesteatoma specimens were obtained from patients undergoing middle ear and mastoid surgery. All of the cholesteatoma specimens were removed from the eroded bone surface. Tissue was frozen immediately and kept in a deep freezer until further use. In preparation for transmission electron microscopy, specimens were fixed for 1.5 h in 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1M cacodylate buffer solution, pH 7.47.6. After a thorough washing in 0.1M cacodylate buffer containing 7% sucrose, tissue was post-fixed for 1.5 h in 1% osmium tetroxide in 0.1M cacodylate buffer. After dehydration, the specimens were embedded in epoxy resins. Ultrathin sections (7080 nm thick) were made and staining completed, after which tissues were observed under the transmission electron microscope. The ultrastructures of LC such as Golgi apparatus, mitochondria and vacuoles were observed after identifying cells by the presence of Birbeck's granules. The apposition phenomenon of Langerhans cell with lymphocyte-like cells was found to give further evidence for a cell-mediated immune response mechanism in the pathogenesis of cholesteatoma.
Preparation of L C LC were enriched using a modification of the method described by Witmer-Pack et al. [22]. In brief, skin was removed from neonatal rats and incubated for 20h in 0.25% trypsin at 4°C. The epidermis was then peeled off and shaken in Ham's F-10 medium. The precipitated cell pellets were washed three times with F-10 medium and resuspended in F-10 medium supplemented with 5% fetal bovine serum (FBS). The cells were placed in Falcon 6-well culture plates (3.7 x 106cells/well) and incubated for 3 days under a humidified chamber with 5% CO2 - 95% air at 37°C. The supernatant containing LC was next obtained and centrifuged at 200g for 12rain. The cell pellets were resuspended with F-10 + 1% FBS in 4.5ml solution. To kill keratinocytes, monoclonal antibody antiThy-1 (Pel-Freez, Rogers, Ark.), 1:50 diluted, and 0.5 ml guinea pig complement (Pel-Freez) were added and incubated at 37°C for 1 h. To remove dead cells, the cellular suspension was diluted with Hank's balanced salt solution (HBSS) (Gibco, Grand Island, N.Y.) and centrifuged. Cell pellets were resuspended in 3ml
381
Fig. 1A, B. Identification of Langerhans cells after enrichment by using mouse anti-rat Ia monoclonal antibody. A Indirect immnnofluorescent stain. B Immunoperoxidase stain. Original magnification, × 200 HBSS, 2 ml 0.25% trypsin and DNase (80 ~tg/ml) (Sigma, St. Louis, Mo.) were added and incubation was carried out at 37°C for 20 min. The suspension was then further diluted with F-10 + 10% FBS and centrifuged. Cell pellets were next resuspended in F10 + 10% FBS to give a concentration of about 5 × 105cells/ml. Monoclonal antibody against rat leukocyte common antigen (PelFreez) was added (1:30 dilution) to bind LC at 4°C for 30min. Cell pellets were washed twice in F-10 medium and then transferred to a Petri dish which was precoated with goat anti-mouse Ig in phosphate-buffered saline (PBS; 50lag per 35-mm dish). Cells were left in the dish for 30 min at 4°C and then adjusted to room temperature for 10min. Non-attached cells were removed from the in vitro medium with a pipette. The attached LC were eluted by competition with rat Ig, 500 gg/ml in PBS, at 4°C for 45 min. Cells were then resuspended in F-10 supplemented with 10% FBS. Final cell numbers were 3 × 10 4 cells/rat. These LC-enriched cells were identified with mouse anti-rat Ia monoclonal antibody (PelFreez) by indirect immunofluorescent and immunoperoxidase staining methods (Fig. 1).
LC-conditioned medium (LCCM) LC were cultured in F-10 medium in a humidified chamber with 5% CO2 - 95% air at 37°C. The conditioned medium was harvested after 48 h incubation. The protein content in LCCM was determined by Lowry's method [10].
Incorporation of 3H-leucine and 3H-putrescine Basal cells were prepared from the skin of neonatal rats [1]. Basal cells (1 × 106cells/ml) were then cultured in serum-free F-10 medium and various concentrations of LCCM (0, 35, 70, 105, 140 gg/ml). To each well, 0.5 gCi/ml 3H-leucine or 3H-putrescine (Dupont, Boston, Mass.) was added. After 24h incubation, cells were harvested and processed for determining the incorporation of 3H-leucine and 3H-putrescine by using a Beckmann liquid scintillation counter.
Results L C were f o u n d either in the epithelial area (Fig. 2) or in the subepithelial area (Fig. 3) o f the h u m a n middle ear
cholesteatomas, using Birbeck's granules as the hallm a r k for the L C (Figs. 3,4). A p p o s i t i o n of the L C with lymphocyte-like cells was f o u n d in the subepithelial connective tissue of the middle ear c h o l e s t e a t o m a s (Fig. 3). L C C M was f o u n d to p r o m o t e the i n c o r p o r a t i o n of 3 H-leucine (Fig. 5) and 3 H-putrescine (Fig. 6) into basal cells as the c o n c e n t r a t i o n of L C C M increased. This finding was considered to show that L C C M stimulated protein synthesis and e n v e l o p e f o r m a t i o n during differentiation of basal cells.
Discussion E p i d e r m a l L C are derived f r o m cells originating in b o n e m a r r o w [6]. L C have b e e n f o u n d in m o s t keratinizing stratified s q u a m o u s epithelia, including the external ear canal and the infected t y m p a n i c m e m b r a n e [11], but not in n o r m a l h u m a n middle ear m u c o s a [7] and the n o r m a l t y m p a n i c m e m b r a n e [21]. Recently, epidermal L C have b e e n p r o v e d to be i m m u n o c o m p e t e n t cells which activate cell-mediated i m m u n o l o g i c m e c h a n i s m s [12, 15, 16]. Following their identification in aural c h o l e s t e a t o m a [8], L C were also assumed to be i m p o r t a n t in the regulation of acquired c h o l e s t e a t o m a s [3, 20]. T h e n o r m a l middle ear is a unique mucosal c o m p a r t m e n t which contains very few resident l y m p h o c y t e s and no l y m p h o i d tissue [18]. T h e middle ear is seeded non-specifically by circulating l y m p h o c y t e s in response to antigenic stimulation [13]. As a c o n s e q u e n c e , the expression of cell surface antigens on L C not only serves for identification, but also gives i m p o r t a n t insights into their function. T w o of the antigens expressed by L C in m a n are products of the h u m a n histocompatibility gene ( H L A complex) on the sixth c h r o m o s o m e . This gene encodes two classes of protein that regulate i m m u n e responses to foreign antigens. L C , as well as o t h e r i m m u n o c o m p e t e n t cells, pos-
382
Fig. 2. Two Langerhans cells (LC) in the epithelial layer of a human middle ear cholesteatoma. K, Keratin layer; B, basement membrane. × 4000
Fig. 3. Apposition of Langerhans cell (LC) with a lymphocyte-like cell (L) in the subepithelial connective tissue area of a human middle ear cholesteatoma. Arrow in boxed area points to rod-shaped Birbeck's granules. × 7000
383
Fig. 4. Langerhans cell with Birbeck's granules (arrow) in a human middle ear cholesteatoma. Many mitochondria (M), Golgi apparatus (G) and vacuoles (V) are noted. × 17000 80
0
20
0 X x 13_ 0
60
15
{
__o 10
40
E
"5
"5
Q
p,
z0
0
8
0
0 o mc
0
50
1 O0
150
LC conditioned medium concentration (gg/ml)
0
50
1O0
150
LC conditioned medium concentration (~g/ml)
Fig. 5. Incorporation of 3H-leucine into basal cells cultured in various concentrations of Langerhans cells conditioned medium
Fig. 6. Incorporation of ~H-putrescine into basal cells cultured in various concentrations of Langerhans cells conditioned medium
sess threee antigens, H L A - D R , -DQ and -DP, that participate in the presentation of foreign antigens to helper T-lymphocytes [2]. Their relationship is assumed to be important in regulation of acquired aural cholesteatoma [3, 4, 20, 21]. In this study we used transmission electron microscopy to localize LC in human cholesteatomas (Figs. 2, 3). LC were noted to be present in the epithelial layer and subepithelial connective tissue adjacent to the basement membrane.
Increased numbers of LC in cholesteatomas have been noted for many years [8, 19] and are speculated to have a role in immunobiology. However, the LC of cholesteatoma were found to be different from those in the normal ear canal and tympanic membrane on the basis of an increased Golgi apparatus, lysosomes and a prominent endoplasmic reticulum. Our study localized LC in cholesteatomas, as shown in Fig. 4. These changes suggested increased secretory activity, as reported by other investigators [4]. The p h e n o m e n o n of close apposition of
384 LC with lymphocyte-like cells within the epithelium has also been noted in contact hypersensitivity [16]. A similar condition has been observed in the cholesteatoma matrix [4] and was confirmed by our present studies. These observations suggest a similar immunologic role for LC in cholesteatoma and in contact hypersensitivity reactions involving skin. Incorporation of isotopic leucine can be used as a method for measuring protein synthesis [9], while incorporation of 3H-putrescine into a-casein has been used as a method for detecting transglutaminase activity [5, 14]. Protein synthesis is essential either in the proliferation or differentiation of cholesteatoma basal cells. Since the activity of transglutaminase can be correlated to the terminal differentiation of basal cells, a higher percentage of incorporation of putrescine indicates much more activity in differentiation [5]. Our present study used L C C M collected from the LC culture medium into which certain factors were secreted. The promotion of both protein synthesis of basal cells and envelope formation by LCCM during terminal differentiation in vitro showed another function of L C in addition to its transferring action through direct contact with T-lymphocytes. Our results are in agreement with the observation of Smolle et al. [17], who showed some association of LC with epithelial differentiation. In their report, the LC density in seborrheic keratosis was significantly higher than that in basal cell and squamous cell carcinomas. Our results may also account for the findings of Veldman et al. [20] about the more aggressive behavior of cholesteatomas in young children and in those cases in which extensive residual or recurrent disease was found in revision surgery performed within a year. A n abundant LC density was found in these specimens. In contrast, Palva and Taskinen [11] described LC in the secretory epithelium of the middle ear with secretory otitis media, chronic otitis media and cholesteatoma, and were unable to correlate LC numbers with keratinization or the possible reformation of cholesteatoma. Our present findings of increased LC activity in cholesteatomas and the apposition of LC with lymphocytelike cells suggest an important role in the clinical pathogenesis of cholesteatoma. LC depletion or immunosuppression is now being used for the treatment of various cell-mediated skin diseases. With a better understanding of cellular interactions resulting in the development and recurrence of aural cholesteatoma, new insights in treatment may be achieved by controlling the cellular interactions taking place. Since cells extracted from rat skin may have a different response from that of cells from human middle ear cholesteatoma, further investigations are necessary to compare the biological roles of both tissues. Acknowledgements. This investigation was supported by grants from NSC 79-0412-B006-28, Taiwan.
References 1. Chao WY, Sugita T, Huang CC (1989) Fibronectin in middle ear cholesteatoma. In: Tos M, Thomsen J, Peitersen E (eds) Cholesteatoma and mastoid surgery. Kugler and Ghedini, Amsterdam, pp 129-134
2. Cruchley AT, Williams DM, Farthing PM, Lesch CA, Squier C (1989) Regional variation in Langerhans cell distribution and density in normal human oral mucosa determined using monoclonal antibodies against CD1, HLADR, HLADQ and HLADP. J Oral Pathol Med 18 : 510-516 3. Gantz BJ (1984) Epidermal Langerhans cells in cholesteatoma. Ann Otol Rhinol Laryngol 93 : 150-156 4. Gantz BJ, Hart MJ (1987) Immunology of acquired aural cholesteatoma. In: Berstein JM, Ogra PL (eds) Immunology of the ear. Raven Press, NewYork, pp 391-402 5. Huang CC, Yi ZX, Cbao WY (1988) Effects of granulation tissue conditioned medium on differentiation of keratinocytes. Arch Otorhinolaryngol 245 : 325-329 6. Katz SI, Tamaki K, Sachs DH (1979) Epidermal Langerhans cells are derived from cells originating in bone marrow. Nature 282 : 324-326 7. Kawabata I, Paparella MM (1969) Ultrastructure of normal human middle ear mocosa. Ann Otol Rhinol Laryngol 78: 125-137 8. Lira D J, Saunders WH (1972) Acquired cholesteatoma. Light and electron microscopicobservations. Ann Otol Rhinol Laryngol 81:2-12 9. Low RB, Hildebran JN, Absher PM, Stirewalt WS, Arnold J (1986) Comparison of the use of isotopic proline vs leucine to measure protein synthesis in cultured fibroblasts. Connect Tissue Res 14:179-185 10. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193 : 265-275 11. Palva T, Taskinen E (1987) Inflammatory cell subpopulations in chronic otitis media. The Langerhans' cells. Arch Otolaryngol Head Neck Surg 113 : 149-154 12. Pehamberger H, Stingl LA, Pogantsch S, Steiner G, Wolff K, Stingl G (1983) Epidermal cell-induced generation of cytotoxic T-lymphocyte responses against alloantigens or TNP-modified syngeneic cells: requirement for Ia-positive Langerhans cells. J Invest Dermatol 81:208-211 13. Ryan AF, Sharp PA, Harris JP (1990) Lymphocyte circulation to the middle ear. Acta Otolaryngol (Stockh) 109 : 278-287 14. Schroff G, Neumann C, Sorg C (1981) Transglutaminase as a marker for subsets of routine macrophages. Eur J Immunol 11 : 637-642 15. Shiohara T, Moriya N, Saizawa KM, Nagashima M (1988) Role of Langerhans cells in epidermotropism of T cells. Arch Dermatol Res 280 : 33-38 16. Silberberg I (1973) Apposition of mononuclear cells to Langerhans cells in contact allergic reactions. Acta Dermatovenereol (Stockh) 53 : 1-12 17. Smolle J, Soyer HP, Ehall R, Bartenstein S, Kerl H (1986) Langerhans cells density in epithelial skin tumors correlates with epithelial differentiation but not with the peritumoral infiltrate. J Invest Dermatol 87 : 477-479 18. Takahashi M, Peppard J, Harris JP (1989) Immunohistochemical study of the murine middle ear and Eustachian tube. Acta Otolaryngol (Stockh) 107:97-103 19. Takahashi S, Nakano Y (1989) Immunohistochemical demonstration of Langerhans' cell in cholesteatoma using an antiserum against S-100 protein. Arch Otorhinolaryngol 246:4852 20. Veldman JE, Visser CE, Schuurman HJ, Groot JCMJ de, Huizing EH (1984) Immunobiology of Langerhans' cells migrating into aural cholesteatomas. Otolaryngol Head Neck Surg 92 : 1-6 21. Veldman JE, Dijk CM van, Visser CE, Huizing EH (1987) Aural cholesteatoma and immunity. In: Veldman JE, McCabe BF (eds) Otoimmunology. Kugler, Amsterdam, pp 69-79 22. Witmer-Pack MD, Olivier W, Valinsky J, Schuler G, Steinman RM (1987) Granulocyte/macrophage colony-stimulating factor is essential for the viability and function of cultured murine epidermal Langerhans cells. J Exp Med 166:14841498
European Archives of
Oto-RhinoLaryngology © Springer-Verlag 1992
Langerhans cells in human middle ear cholesteatomas W.-Y. Chao ~, Y.-T. Jin 2, and C.-C. Huang 3 Departments of 10tolaryngology and 2Pathology, National Cheng Kung University College of Medicine, Tainan, 70101 Taiwan 3Department of Otolaryngology, Columbia University College of Physicians and Surgeons, NewYork, NY 10032, USA Received January 21, 1992 / Accepted June 16, 1992
Summary. Langerhans cells have been found in cholesteatomas for many years. It is believed that they are immunocompetent cells and have the same role in cellmediated immunologic mechanisms in cholesteatoma as well as in skin. This study used the transmission electron microscope to observe the cellular characteristics of Langerhans cells and the apposition p h e n o m e n o n of Langerhans cells with lymphocyte-like cells in human middle ear cholesteatomatous tissue. These findings are evidence for cell-mediated immune responses in middle ear cholesteatomas. In vitro Langerhans cells conditioned medium prepared from Lewis rat skin was used to show its effects on protein synthesis and the differentiation of basal cells. Since the cellular behaviour of basal cells is important in the development and pathogenesis of cholesteatoma, the present study shows that Langerhans cells may have some role in the clinical formation of a cholesteatoma. Since cells extracted from rat skin may have a different response from that of cells from human middle ear cholesteatoma, further investigations are necessary to compare the biological effects of both tissues. Key words: Cholesteatoma - Langerhans cells - Basal cells - T-lymphocytes
Introduction Cholesteatoma is a c o m m o n middle ear disease which is pathologically benign but clinically destructive in the temporal bone. It is composed of epithelial matrix and connective tissue, including granulation tissue and many inflammatory cells. The roles of cellular components in the pathogenesis of cholesteatoma have been studied by many institutes. A m o n g them, Langerhans cells (LC) have been noted for many years, but their relationship with T-lymphocytes has been assumed to be clinically important in the disease process only recently. In the present study transmission electron microscopy was used to study human middle ear cholesteatomas obtained from ear surgery to provide some explanation for the pathogenesis of this disease. Previous studies have shown that LC density in seborrheic keratosis is significantly higher than that in basal cell and squamous cell carcino-
Correspondence to: W.-Y. Chao
mas [4]. To determine whether LC plays a role in promoting differentiation of the epithelial cells of a cholesteatoma, this study was designed to demonstrate that the in vitro cellular behavior of basal cells is influenced by the factors released from LC into the tissue medium. To date, the treatment of a temporal bone cholesteatoma has been surgical removal. Understanding the roles of various cells in cholesteatomas may provide some insight for future management.
Materials and methods Sixteen human middle ear cholesteatoma specimens were obtained from patients undergoing middle ear and mastoid surgery. All of the cholesteatoma specimens were removed from the eroded bone surface. Tissue was frozen immediately and kept in a deep freezer until further use. In preparation for transmission electron microscopy, specimens were fixed for 1.5 h in 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1M cacodylate buffer solution, pH 7.47.6. After a thorough washing in 0.1M cacodylate buffer containing 7% sucrose, tissue was post-fixed for 1.5 h in 1% osmium tetroxide in 0.1M cacodylate buffer. After dehydration, the specimens were embedded in epoxy resins. Ultrathin sections (7080 nm thick) were made and staining completed, after which tissues were observed under the transmission electron microscope. The ultrastructures of LC such as Golgi apparatus, mitochondria and vacuoles were observed after identifying cells by the presence of Birbeck's granules. The apposition phenomenon of Langerhans cell with lymphocyte-like cells was found to give further evidence for a cell-mediated immune response mechanism in the pathogenesis of cholesteatoma.
Preparation of L C LC were enriched using a modification of the method described by Witmer-Pack et al. [22]. In brief, skin was removed from neonatal rats and incubated for 20h in 0.25% trypsin at 4°C. The epidermis was then peeled off and shaken in Ham's F-10 medium. The precipitated cell pellets were washed three times with F-10 medium and resuspended in F-10 medium supplemented with 5% fetal bovine serum (FBS). The cells were placed in Falcon 6-well culture plates (3.7 x 106cells/well) and incubated for 3 days under a humidified chamber with 5% CO2 - 95% air at 37°C. The supernatant containing LC was next obtained and centrifuged at 200g for 12rain. The cell pellets were resuspended with F-10 + 1% FBS in 4.5ml solution. To kill keratinocytes, monoclonal antibody antiThy-1 (Pel-Freez, Rogers, Ark.), 1:50 diluted, and 0.5 ml guinea pig complement (Pel-Freez) were added and incubated at 37°C for 1 h. To remove dead cells, the cellular suspension was diluted with Hank's balanced salt solution (HBSS) (Gibco, Grand Island, N.Y.) and centrifuged. Cell pellets were resuspended in 3ml
381
Fig. 1A, B. Identification of Langerhans cells after enrichment by using mouse anti-rat Ia monoclonal antibody. A Indirect immnnofluorescent stain. B Immunoperoxidase stain. Original magnification, × 200 HBSS, 2 ml 0.25% trypsin and DNase (80 ~tg/ml) (Sigma, St. Louis, Mo.) were added and incubation was carried out at 37°C for 20 min. The suspension was then further diluted with F-10 + 10% FBS and centrifuged. Cell pellets were next resuspended in F10 + 10% FBS to give a concentration of about 5 × 105cells/ml. Monoclonal antibody against rat leukocyte common antigen (PelFreez) was added (1:30 dilution) to bind LC at 4°C for 30min. Cell pellets were washed twice in F-10 medium and then transferred to a Petri dish which was precoated with goat anti-mouse Ig in phosphate-buffered saline (PBS; 50lag per 35-mm dish). Cells were left in the dish for 30 min at 4°C and then adjusted to room temperature for 10min. Non-attached cells were removed from the in vitro medium with a pipette. The attached LC were eluted by competition with rat Ig, 500 gg/ml in PBS, at 4°C for 45 min. Cells were then resuspended in F-10 supplemented with 10% FBS. Final cell numbers were 3 × 10 4 cells/rat. These LC-enriched cells were identified with mouse anti-rat Ia monoclonal antibody (PelFreez) by indirect immunofluorescent and immunoperoxidase staining methods (Fig. 1).
LC-conditioned medium (LCCM) LC were cultured in F-10 medium in a humidified chamber with 5% CO2 - 95% air at 37°C. The conditioned medium was harvested after 48 h incubation. The protein content in LCCM was determined by Lowry's method [10].
Incorporation of 3H-leucine and 3H-putrescine Basal cells were prepared from the skin of neonatal rats [1]. Basal cells (1 × 106cells/ml) were then cultured in serum-free F-10 medium and various concentrations of LCCM (0, 35, 70, 105, 140 gg/ml). To each well, 0.5 gCi/ml 3H-leucine or 3H-putrescine (Dupont, Boston, Mass.) was added. After 24h incubation, cells were harvested and processed for determining the incorporation of 3H-leucine and 3H-putrescine by using a Beckmann liquid scintillation counter.
Results L C were f o u n d either in the epithelial area (Fig. 2) or in the subepithelial area (Fig. 3) o f the h u m a n middle ear
cholesteatomas, using Birbeck's granules as the hallm a r k for the L C (Figs. 3,4). A p p o s i t i o n of the L C with lymphocyte-like cells was f o u n d in the subepithelial connective tissue of the middle ear c h o l e s t e a t o m a s (Fig. 3). L C C M was f o u n d to p r o m o t e the i n c o r p o r a t i o n of 3 H-leucine (Fig. 5) and 3 H-putrescine (Fig. 6) into basal cells as the c o n c e n t r a t i o n of L C C M increased. This finding was considered to show that L C C M stimulated protein synthesis and e n v e l o p e f o r m a t i o n during differentiation of basal cells.
Discussion E p i d e r m a l L C are derived f r o m cells originating in b o n e m a r r o w [6]. L C have b e e n f o u n d in m o s t keratinizing stratified s q u a m o u s epithelia, including the external ear canal and the infected t y m p a n i c m e m b r a n e [11], but not in n o r m a l h u m a n middle ear m u c o s a [7] and the n o r m a l t y m p a n i c m e m b r a n e [21]. Recently, epidermal L C have b e e n p r o v e d to be i m m u n o c o m p e t e n t cells which activate cell-mediated i m m u n o l o g i c m e c h a n i s m s [12, 15, 16]. Following their identification in aural c h o l e s t e a t o m a [8], L C were also assumed to be i m p o r t a n t in the regulation of acquired c h o l e s t e a t o m a s [3, 20]. T h e n o r m a l middle ear is a unique mucosal c o m p a r t m e n t which contains very few resident l y m p h o c y t e s and no l y m p h o i d tissue [18]. T h e middle ear is seeded non-specifically by circulating l y m p h o c y t e s in response to antigenic stimulation [13]. As a c o n s e q u e n c e , the expression of cell surface antigens on L C not only serves for identification, but also gives i m p o r t a n t insights into their function. T w o of the antigens expressed by L C in m a n are products of the h u m a n histocompatibility gene ( H L A complex) on the sixth c h r o m o s o m e . This gene encodes two classes of protein that regulate i m m u n e responses to foreign antigens. L C , as well as o t h e r i m m u n o c o m p e t e n t cells, pos-
382
Fig. 2. Two Langerhans cells (LC) in the epithelial layer of a human middle ear cholesteatoma. K, Keratin layer; B, basement membrane. × 4000
Fig. 3. Apposition of Langerhans cell (LC) with a lymphocyte-like cell (L) in the subepithelial connective tissue area of a human middle ear cholesteatoma. Arrow in boxed area points to rod-shaped Birbeck's granules. × 7000
383
Fig. 4. Langerhans cell with Birbeck's granules (arrow) in a human middle ear cholesteatoma. Many mitochondria (M), Golgi apparatus (G) and vacuoles (V) are noted. × 17000 80
0
20
0 X x 13_ 0
60
15
{
__o 10
40
E
"5
"5
Q
p,
z0
0
8
0
0 o mc
0
50
1 O0
150
LC conditioned medium concentration (gg/ml)
0
50
1O0
150
LC conditioned medium concentration (~g/ml)
Fig. 5. Incorporation of 3H-leucine into basal cells cultured in various concentrations of Langerhans cells conditioned medium
Fig. 6. Incorporation of ~H-putrescine into basal cells cultured in various concentrations of Langerhans cells conditioned medium
sess threee antigens, H L A - D R , -DQ and -DP, that participate in the presentation of foreign antigens to helper T-lymphocytes [2]. Their relationship is assumed to be important in regulation of acquired aural cholesteatoma [3, 4, 20, 21]. In this study we used transmission electron microscopy to localize LC in human cholesteatomas (Figs. 2, 3). LC were noted to be present in the epithelial layer and subepithelial connective tissue adjacent to the basement membrane.
Increased numbers of LC in cholesteatomas have been noted for many years [8, 19] and are speculated to have a role in immunobiology. However, the LC of cholesteatoma were found to be different from those in the normal ear canal and tympanic membrane on the basis of an increased Golgi apparatus, lysosomes and a prominent endoplasmic reticulum. Our study localized LC in cholesteatomas, as shown in Fig. 4. These changes suggested increased secretory activity, as reported by other investigators [4]. The p h e n o m e n o n of close apposition of
384 LC with lymphocyte-like cells within the epithelium has also been noted in contact hypersensitivity [16]. A similar condition has been observed in the cholesteatoma matrix [4] and was confirmed by our present studies. These observations suggest a similar immunologic role for LC in cholesteatoma and in contact hypersensitivity reactions involving skin. Incorporation of isotopic leucine can be used as a method for measuring protein synthesis [9], while incorporation of 3H-putrescine into a-casein has been used as a method for detecting transglutaminase activity [5, 14]. Protein synthesis is essential either in the proliferation or differentiation of cholesteatoma basal cells. Since the activity of transglutaminase can be correlated to the terminal differentiation of basal cells, a higher percentage of incorporation of putrescine indicates much more activity in differentiation [5]. Our present study used L C C M collected from the LC culture medium into which certain factors were secreted. The promotion of both protein synthesis of basal cells and envelope formation by LCCM during terminal differentiation in vitro showed another function of L C in addition to its transferring action through direct contact with T-lymphocytes. Our results are in agreement with the observation of Smolle et al. [17], who showed some association of LC with epithelial differentiation. In their report, the LC density in seborrheic keratosis was significantly higher than that in basal cell and squamous cell carcinomas. Our results may also account for the findings of Veldman et al. [20] about the more aggressive behavior of cholesteatomas in young children and in those cases in which extensive residual or recurrent disease was found in revision surgery performed within a year. A n abundant LC density was found in these specimens. In contrast, Palva and Taskinen [11] described LC in the secretory epithelium of the middle ear with secretory otitis media, chronic otitis media and cholesteatoma, and were unable to correlate LC numbers with keratinization or the possible reformation of cholesteatoma. Our present findings of increased LC activity in cholesteatomas and the apposition of LC with lymphocytelike cells suggest an important role in the clinical pathogenesis of cholesteatoma. LC depletion or immunosuppression is now being used for the treatment of various cell-mediated skin diseases. With a better understanding of cellular interactions resulting in the development and recurrence of aural cholesteatoma, new insights in treatment may be achieved by controlling the cellular interactions taking place. Since cells extracted from rat skin may have a different response from that of cells from human middle ear cholesteatoma, further investigations are necessary to compare the biological roles of both tissues. Acknowledgements. This investigation was supported by grants from NSC 79-0412-B006-28, Taiwan.
References 1. Chao WY, Sugita T, Huang CC (1989) Fibronectin in middle ear cholesteatoma. In: Tos M, Thomsen J, Peitersen E (eds) Cholesteatoma and mastoid surgery. Kugler and Ghedini, Amsterdam, pp 129-134
2. Cruchley AT, Williams DM, Farthing PM, Lesch CA, Squier C (1989) Regional variation in Langerhans cell distribution and density in normal human oral mucosa determined using monoclonal antibodies against CD1, HLADR, HLADQ and HLADP. J Oral Pathol Med 18 : 510-516 3. Gantz BJ (1984) Epidermal Langerhans cells in cholesteatoma. Ann Otol Rhinol Laryngol 93 : 150-156 4. Gantz BJ, Hart MJ (1987) Immunology of acquired aural cholesteatoma. In: Berstein JM, Ogra PL (eds) Immunology of the ear. Raven Press, NewYork, pp 391-402 5. Huang CC, Yi ZX, Cbao WY (1988) Effects of granulation tissue conditioned medium on differentiation of keratinocytes. Arch Otorhinolaryngol 245 : 325-329 6. Katz SI, Tamaki K, Sachs DH (1979) Epidermal Langerhans cells are derived from cells originating in bone marrow. Nature 282 : 324-326 7. Kawabata I, Paparella MM (1969) Ultrastructure of normal human middle ear mocosa. Ann Otol Rhinol Laryngol 78: 125-137 8. Lira D J, Saunders WH (1972) Acquired cholesteatoma. Light and electron microscopicobservations. Ann Otol Rhinol Laryngol 81:2-12 9. Low RB, Hildebran JN, Absher PM, Stirewalt WS, Arnold J (1986) Comparison of the use of isotopic proline vs leucine to measure protein synthesis in cultured fibroblasts. Connect Tissue Res 14:179-185 10. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193 : 265-275 11. Palva T, Taskinen E (1987) Inflammatory cell subpopulations in chronic otitis media. The Langerhans' cells. Arch Otolaryngol Head Neck Surg 113 : 149-154 12. Pehamberger H, Stingl LA, Pogantsch S, Steiner G, Wolff K, Stingl G (1983) Epidermal cell-induced generation of cytotoxic T-lymphocyte responses against alloantigens or TNP-modified syngeneic cells: requirement for Ia-positive Langerhans cells. J Invest Dermatol 81:208-211 13. Ryan AF, Sharp PA, Harris JP (1990) Lymphocyte circulation to the middle ear. Acta Otolaryngol (Stockh) 109 : 278-287 14. Schroff G, Neumann C, Sorg C (1981) Transglutaminase as a marker for subsets of routine macrophages. Eur J Immunol 11 : 637-642 15. Shiohara T, Moriya N, Saizawa KM, Nagashima M (1988) Role of Langerhans cells in epidermotropism of T cells. Arch Dermatol Res 280 : 33-38 16. Silberberg I (1973) Apposition of mononuclear cells to Langerhans cells in contact allergic reactions. Acta Dermatovenereol (Stockh) 53 : 1-12 17. Smolle J, Soyer HP, Ehall R, Bartenstein S, Kerl H (1986) Langerhans cells density in epithelial skin tumors correlates with epithelial differentiation but not with the peritumoral infiltrate. J Invest Dermatol 87 : 477-479 18. Takahashi M, Peppard J, Harris JP (1989) Immunohistochemical study of the murine middle ear and Eustachian tube. Acta Otolaryngol (Stockh) 107:97-103 19. Takahashi S, Nakano Y (1989) Immunohistochemical demonstration of Langerhans' cell in cholesteatoma using an antiserum against S-100 protein. Arch Otorhinolaryngol 246:4852 20. Veldman JE, Visser CE, Schuurman HJ, Groot JCMJ de, Huizing EH (1984) Immunobiology of Langerhans' cells migrating into aural cholesteatomas. Otolaryngol Head Neck Surg 92 : 1-6 21. Veldman JE, Dijk CM van, Visser CE, Huizing EH (1987) Aural cholesteatoma and immunity. In: Veldman JE, McCabe BF (eds) Otoimmunology. Kugler, Amsterdam, pp 69-79 22. Witmer-Pack MD, Olivier W, Valinsky J, Schuler G, Steinman RM (1987) Granulocyte/macrophage colony-stimulating factor is essential for the viability and function of cultured murine epidermal Langerhans cells. J Exp Med 166:14841498
