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CD1a-Positive Cells in Odontogenic Cysts
JOURNAL OF ENDODONTICS Copyright © 2002 by The American Association of Endodontists
Printed in U.S.A. VOL. 28, NO. 4, APRIL 2002
SCIENTIFIC ARTICLES CD1a-Positive Cells in Odontogenic Cysts Adriano Piattelli, MD, DDS, Corrado Rubini, MD, Giovanna Iezzi, DDS, and Massimiliano Fioroni, DDS
MATERIALS AND METHODS
Langerhans cells (LC) are bone marrow-derived cells that have a CD1a-positive immunophenotype and are an important portion of the cellmediated immune response. The aim of this study was an immunohistochemical evaluation of CD1a positive cells in different types of oral cysts. Fifty-five cysts were studied: 18 odontogenic keratocysts (OKC), of which five were orthokeratotic and 13 parakeratotic; 19 radicular cysts; and 18 dentigerous cysts. Positive LC was 80% for orthokeratotic OKC, 33% for parakeratotic OKC, approximately 35% for radicular cysts, and approximately 20% for dentigerous cysts. The results show that OKC with well-differentiated epithelial linings presented a greater number of LC than the other cysts. However, when the cyst wall was inflamed there were no differences in LC expression in the different types of cysts. The data confirm that LC distribution seems to be associated with the degree of differentiation of the epithelia.
Fifty-five cysts were examined: 18 odontogenic keratocysts (OKC), of which five were orthokeratotic and 13 parakeratotic; 19 radicular cysts (RC); and 18 dentigerous cysts (DC). The age of the patients ranged from 19 to 59 yr (mean ⫽ 43 yr). All specimens had been routinely fixed in 10% neutral buffered formalin (24 to 48 h), dehydrated in graded alcohols, cleared in xylene, and embedded in paraffin. The hematoxylin-eosin stained slides were all reviewed, the diagnoses confirmed, and slides for quantitative evaluation selected. For CD1a immunostaining, the slides were pretreated with 3-aminopropyltriethoxysilane (APES; Sigma Chemical Co., St. Louis, MO), which avoided the separation of the section from the slide during incubation in the microwave oven. For each case, a 5-micron section was cut and placed on a pretreated slide. The slide staining protocol consisted of the application of a series of reagents as follows: • Dry overnight at 37°C; • Dewax and rehydrate; • Immerse in a plastic box containing 0.01 M citrate buffer at pH 6.0; • Incubate for 5 min in a microwave oven, initially at 750 watts until boiling begins and then at 350 watts for the remaining time; • Incubate for 5 min in a microwave oven at 350 watts; • Cool for 20 min at room temperature; • Wash in running water and then in distilled water for 5 min; • Wash in Tris-buffer saline (TBS) for 5 min; • Remove excess TBS; • Add anti-CD1a (prediluted) antibodies (Immunotech Inc., Westbrook, ME) in TBS; • Incubate for 60 min at room temperature; • Wash in TBS for 5 min (3 times); • Add secondary prediluted biotinylated anti-mouse antibody (LSAB-DAKO; DAKO, Glostrup, Denmark) and incubate for 10 min at room temperature; • Wash in TBS for 5 min (3 times); • Add prediluted streptavidin-peroxidase complex (DAKO) and incubate for 10 min at room temperature; • Wash in TBS for 5 min (3 times);
Langerhans cells (LC) are bone marrow-derived dendritic cells that have a CD1a-positive immunophenotype (1–5). LC are an important component of the cell-mediated immune response (2). LC migrate from the bone marrow to the epithelium (4). Epidermal LC constitute approximately 1% to 4% of the epidermal cells (1, 4). LC are usually located in the suprabasal and spinous layers of the oral gingival and sulcular epithelia (2– 6). LC are believed to play a significant role in tumor immunology, having a function against the emergence of new antigens expressed by malignant transformation (4, 7, 8) and may have a role in the development of gingival inflammation (3, 4). Both humoral and cell-mediated immunological reactions have been proposed in the pathogenesis of oral cysts (4). The aim of this study was an immunohistochemical evaluation of CD1a-positive cells in different types of oral cysts. 267
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FIG 1. Parakeratotic OKC. Positive LC can be seen in the parabasal layer (arrow) (Alkaline Phosphatase Anti-Alkaline Phosphatase ⫻40).
• Immerse in 0.05% DAB and 0.01% H2O2 in TBS for 2 to 3 min at room temperature; • Wash in running water and then in distilled water for 5 min; • Counterstain with ethyl green for 30 min; • Wash in distilled water for 30 s; • Wash in butanol I for 5 s; • Wash in butanol II for 30 s; • Dehydrate and mount in Permount. The positivity to CD1a was evaluated by counting the number of positive cells in 1000 cells, and the values were expressed as a percentage. RESULTS In three of the orthokeratotic OKC there was a strong positivity of LC, which were located in the basal (3%) and parabasal (9%) layers, similar to normal oral mucosa. In one orthokeratotic OKC there was only mild parabasal positivity (1%), whereas the remaining cyst was negative. Positive LC was 80% in orthokeratotic OKC. Four of the parakeratotic OKC (Fig. 1) showed complete negativity for LC in the basal layers and a mild positivity in the parabasal layers (1%), whereas the epithelium was regular and the inflammatory infiltrate was absent. In the areas in which it was possible to find an inflammatory infiltrate and where the epithelium was acanthotic, it was possible to observe positive LC— almost exclusively in the parabasal layers (9%). In eight of the remaining parakeratotic OKC (two were recurrences), LC was completely negative. Positive LC was 33% in parakeratotic OKC. Six RC were positive only in parabasal-superficial layers (3%), where there was an acanthotic epithelium. In areas with a thin epithelium, no positivity was present. Positive LC in RC was approximately 35%. Positive LC (3%) were present in 4 of 18 DC, particularly in the parabasal-superficial layers. Positive LC in DC was approximately 20%. DISCUSSION LC have important antigen-presenting roles in both normal and pathological epidermis and oral epithelium (9). RC are believed to be inflammatory lesions resulting from continuous antigenic stim-
ulation from the root canal (9). The large number of immune cells in periapical granulomas and RC suggests that immunological reactions are important in the development of these lesions and in the epithelial proliferation (4, 9). It is, therefore, conceivable that LC may have an important role in the initiation of an immune reaction associated with cyst formation (4, 9). The tropism of LC for squamous epithelium may explain their presence in the epithelial wall of oral cysts (10). Our results confirm that LC distribution appears to be associated with the degree of differentiation of the epithelia (4). In fact, in orthokeratotic OKC with a well-differentiated epithelium, there was a higher expression of LC, whereas in parakeratotic OKC, where the epithelium matures in an abnormal way, the quantity of LC decreased. On the contrary, Akhlaghi and Dourov (10) found S-100 –positive cells in all cases of odontogenic cysts. Our data were similar to those presented by Gao et al. (9), who reported that OKC with well-differentiated epithelial linings had a greater number of LC than the other cysts. However, in our study, no differences in LC expression were observed in the different types of cysts (OKC, RC, and DC) when the cyst wall was inflamed. On the other hand, Akhlaghi and Dourov (10) found that the number of LC did not always correlate with an inflammatory condition. However, Murase et al. (11) reported that the cases with S-100 protein-positive LC were usually accompanied with a high degree of inflammatory infiltration of the lesions; on the contrary, the negative cases generally lacked inflammatory responses. This work was partially supported by the National Research Council (C.N.R.), Rome, Italy, and by the Ministry of the University, Research, Science and Technology (M.U.R.S.T.), Rome, Italy. Dr. Piattelli is professor, Department of Oral Medicine and Pathology, Dental School, University of Chieti, Chieti, Italy, and honorary senior lecturer, Eastman Dental Institute for Oral Health Care Sciences, London, United Kingdom. Dr. Rubini is researcher, Department of Pathologic Anatomy and Histopathology, University of Ancona, Ancona, Italy. Dr. Iezzi is research fellow, Dental School, University of Chieti, Chieti, Italy. Dr. Fioroni is research fellow, Dental School, University of Ancona, Ancona, Italy. Address requests for reprints to Dr. Adriano Piattelli, Via F. Sciucchi 63, 66100 Chieti, Italy.
References 1. Wright-Browne V, McLain KL, Talpaz M, Ordonez N, Estrov Z. Physiology and pathophysiology of dendritic cells. Hum Pathol 1997;28:563–79. 2. Cruchley AT, Williams DM, Farthing PM, Lesch CA, Squier CA. 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 1989;18:510 – 6. 3. Se´guier S, Godeau G, Brousse N. Immunohistological and morphometric analysis of intraepithelial lymphocytes and Langerhans cells in healthy and diseased human gingival tissues. Arch Oral Biol 2000;45:441–52. 4. Lombardi T, Hauser C, Budtz-Jorgensen E. Langerhans cells: structure, function, and role in oral pathological conditions. J Oral Pathol Med 1993;22: 193–202. 5. Emile JF, Fraitag S, Leborgne M, de Prost Y, Brousse N. Langerhans cell histiocytosis cells are activated Langerhans cells. J Pathol 1994;174: 71– 6. 6. Charbit Y, Monteil RA, Hitzig C, Sanget P, Benaiche N, Jasmin JR. S-100 immunolabeling of Langerhans cells in oral epithelium. J Oral Pathol 1986;15:419 –22. 7. Kurihara K, Hashimoto N. The pathological significance of Langerhans cells in oral cancer. J Oral Pathol 1985;14:289 –93. 8. Pitigala-Arachchi A, Crane IJ, Scully C, Prime SS. Epithelial dendritic cells in pathological human tissues. J Oral Pathol Med 1989;18:11– 6. 9. Gao Z, Mackenzie IC, Rittman BR, Korszun AK, Williams DM, Cruchley AT. Immunocytochemical examination of immune cells in periapical granulomata and odontogenic cysts. J Oral Pathol 1988;17:84 –90. 10. Akhlaghi E, Dourov N. Langerhans cells in odontogenic cysts: a retrospective study based on 142 cases. Bull Group Int Rech Sci Stomatol Odontol 1995;38:71– 6. 11. Murase N, Tatemoto Y, Iwai Y, Okada Y, Mori M. Langerhans cells in odontogenic tumours and cysts as detected by S-100 protein immunohistochemistry. Basic Appl Histochem 1990;34:135– 41.
Printed in U.S.A. VOL. 28, NO. 4, APRIL 2002
SCIENTIFIC ARTICLES CD1a-Positive Cells in Odontogenic Cysts Adriano Piattelli, MD, DDS, Corrado Rubini, MD, Giovanna Iezzi, DDS, and Massimiliano Fioroni, DDS
MATERIALS AND METHODS
Langerhans cells (LC) are bone marrow-derived cells that have a CD1a-positive immunophenotype and are an important portion of the cellmediated immune response. The aim of this study was an immunohistochemical evaluation of CD1a positive cells in different types of oral cysts. Fifty-five cysts were studied: 18 odontogenic keratocysts (OKC), of which five were orthokeratotic and 13 parakeratotic; 19 radicular cysts; and 18 dentigerous cysts. Positive LC was 80% for orthokeratotic OKC, 33% for parakeratotic OKC, approximately 35% for radicular cysts, and approximately 20% for dentigerous cysts. The results show that OKC with well-differentiated epithelial linings presented a greater number of LC than the other cysts. However, when the cyst wall was inflamed there were no differences in LC expression in the different types of cysts. The data confirm that LC distribution seems to be associated with the degree of differentiation of the epithelia.
Fifty-five cysts were examined: 18 odontogenic keratocysts (OKC), of which five were orthokeratotic and 13 parakeratotic; 19 radicular cysts (RC); and 18 dentigerous cysts (DC). The age of the patients ranged from 19 to 59 yr (mean ⫽ 43 yr). All specimens had been routinely fixed in 10% neutral buffered formalin (24 to 48 h), dehydrated in graded alcohols, cleared in xylene, and embedded in paraffin. The hematoxylin-eosin stained slides were all reviewed, the diagnoses confirmed, and slides for quantitative evaluation selected. For CD1a immunostaining, the slides were pretreated with 3-aminopropyltriethoxysilane (APES; Sigma Chemical Co., St. Louis, MO), which avoided the separation of the section from the slide during incubation in the microwave oven. For each case, a 5-micron section was cut and placed on a pretreated slide. The slide staining protocol consisted of the application of a series of reagents as follows: • Dry overnight at 37°C; • Dewax and rehydrate; • Immerse in a plastic box containing 0.01 M citrate buffer at pH 6.0; • Incubate for 5 min in a microwave oven, initially at 750 watts until boiling begins and then at 350 watts for the remaining time; • Incubate for 5 min in a microwave oven at 350 watts; • Cool for 20 min at room temperature; • Wash in running water and then in distilled water for 5 min; • Wash in Tris-buffer saline (TBS) for 5 min; • Remove excess TBS; • Add anti-CD1a (prediluted) antibodies (Immunotech Inc., Westbrook, ME) in TBS; • Incubate for 60 min at room temperature; • Wash in TBS for 5 min (3 times); • Add secondary prediluted biotinylated anti-mouse antibody (LSAB-DAKO; DAKO, Glostrup, Denmark) and incubate for 10 min at room temperature; • Wash in TBS for 5 min (3 times); • Add prediluted streptavidin-peroxidase complex (DAKO) and incubate for 10 min at room temperature; • Wash in TBS for 5 min (3 times);
Langerhans cells (LC) are bone marrow-derived dendritic cells that have a CD1a-positive immunophenotype (1–5). LC are an important component of the cell-mediated immune response (2). LC migrate from the bone marrow to the epithelium (4). Epidermal LC constitute approximately 1% to 4% of the epidermal cells (1, 4). LC are usually located in the suprabasal and spinous layers of the oral gingival and sulcular epithelia (2– 6). LC are believed to play a significant role in tumor immunology, having a function against the emergence of new antigens expressed by malignant transformation (4, 7, 8) and may have a role in the development of gingival inflammation (3, 4). Both humoral and cell-mediated immunological reactions have been proposed in the pathogenesis of oral cysts (4). The aim of this study was an immunohistochemical evaluation of CD1a-positive cells in different types of oral cysts. 267
268
Piattelli et al.
Journal of Endodontics
FIG 1. Parakeratotic OKC. Positive LC can be seen in the parabasal layer (arrow) (Alkaline Phosphatase Anti-Alkaline Phosphatase ⫻40).
• Immerse in 0.05% DAB and 0.01% H2O2 in TBS for 2 to 3 min at room temperature; • Wash in running water and then in distilled water for 5 min; • Counterstain with ethyl green for 30 min; • Wash in distilled water for 30 s; • Wash in butanol I for 5 s; • Wash in butanol II for 30 s; • Dehydrate and mount in Permount. The positivity to CD1a was evaluated by counting the number of positive cells in 1000 cells, and the values were expressed as a percentage. RESULTS In three of the orthokeratotic OKC there was a strong positivity of LC, which were located in the basal (3%) and parabasal (9%) layers, similar to normal oral mucosa. In one orthokeratotic OKC there was only mild parabasal positivity (1%), whereas the remaining cyst was negative. Positive LC was 80% in orthokeratotic OKC. Four of the parakeratotic OKC (Fig. 1) showed complete negativity for LC in the basal layers and a mild positivity in the parabasal layers (1%), whereas the epithelium was regular and the inflammatory infiltrate was absent. In the areas in which it was possible to find an inflammatory infiltrate and where the epithelium was acanthotic, it was possible to observe positive LC— almost exclusively in the parabasal layers (9%). In eight of the remaining parakeratotic OKC (two were recurrences), LC was completely negative. Positive LC was 33% in parakeratotic OKC. Six RC were positive only in parabasal-superficial layers (3%), where there was an acanthotic epithelium. In areas with a thin epithelium, no positivity was present. Positive LC in RC was approximately 35%. Positive LC (3%) were present in 4 of 18 DC, particularly in the parabasal-superficial layers. Positive LC in DC was approximately 20%. DISCUSSION LC have important antigen-presenting roles in both normal and pathological epidermis and oral epithelium (9). RC are believed to be inflammatory lesions resulting from continuous antigenic stim-
ulation from the root canal (9). The large number of immune cells in periapical granulomas and RC suggests that immunological reactions are important in the development of these lesions and in the epithelial proliferation (4, 9). It is, therefore, conceivable that LC may have an important role in the initiation of an immune reaction associated with cyst formation (4, 9). The tropism of LC for squamous epithelium may explain their presence in the epithelial wall of oral cysts (10). Our results confirm that LC distribution appears to be associated with the degree of differentiation of the epithelia (4). In fact, in orthokeratotic OKC with a well-differentiated epithelium, there was a higher expression of LC, whereas in parakeratotic OKC, where the epithelium matures in an abnormal way, the quantity of LC decreased. On the contrary, Akhlaghi and Dourov (10) found S-100 –positive cells in all cases of odontogenic cysts. Our data were similar to those presented by Gao et al. (9), who reported that OKC with well-differentiated epithelial linings had a greater number of LC than the other cysts. However, in our study, no differences in LC expression were observed in the different types of cysts (OKC, RC, and DC) when the cyst wall was inflamed. On the other hand, Akhlaghi and Dourov (10) found that the number of LC did not always correlate with an inflammatory condition. However, Murase et al. (11) reported that the cases with S-100 protein-positive LC were usually accompanied with a high degree of inflammatory infiltration of the lesions; on the contrary, the negative cases generally lacked inflammatory responses. This work was partially supported by the National Research Council (C.N.R.), Rome, Italy, and by the Ministry of the University, Research, Science and Technology (M.U.R.S.T.), Rome, Italy. Dr. Piattelli is professor, Department of Oral Medicine and Pathology, Dental School, University of Chieti, Chieti, Italy, and honorary senior lecturer, Eastman Dental Institute for Oral Health Care Sciences, London, United Kingdom. Dr. Rubini is researcher, Department of Pathologic Anatomy and Histopathology, University of Ancona, Ancona, Italy. Dr. Iezzi is research fellow, Dental School, University of Chieti, Chieti, Italy. Dr. Fioroni is research fellow, Dental School, University of Ancona, Ancona, Italy. Address requests for reprints to Dr. Adriano Piattelli, Via F. Sciucchi 63, 66100 Chieti, Italy.
References 1. Wright-Browne V, McLain KL, Talpaz M, Ordonez N, Estrov Z. Physiology and pathophysiology of dendritic cells. Hum Pathol 1997;28:563–79. 2. Cruchley AT, Williams DM, Farthing PM, Lesch CA, Squier CA. 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 1989;18:510 – 6. 3. Se´guier S, Godeau G, Brousse N. Immunohistological and morphometric analysis of intraepithelial lymphocytes and Langerhans cells in healthy and diseased human gingival tissues. Arch Oral Biol 2000;45:441–52. 4. Lombardi T, Hauser C, Budtz-Jorgensen E. Langerhans cells: structure, function, and role in oral pathological conditions. J Oral Pathol Med 1993;22: 193–202. 5. Emile JF, Fraitag S, Leborgne M, de Prost Y, Brousse N. Langerhans cell histiocytosis cells are activated Langerhans cells. J Pathol 1994;174: 71– 6. 6. Charbit Y, Monteil RA, Hitzig C, Sanget P, Benaiche N, Jasmin JR. S-100 immunolabeling of Langerhans cells in oral epithelium. J Oral Pathol 1986;15:419 –22. 7. Kurihara K, Hashimoto N. The pathological significance of Langerhans cells in oral cancer. J Oral Pathol 1985;14:289 –93. 8. Pitigala-Arachchi A, Crane IJ, Scully C, Prime SS. Epithelial dendritic cells in pathological human tissues. J Oral Pathol Med 1989;18:11– 6. 9. Gao Z, Mackenzie IC, Rittman BR, Korszun AK, Williams DM, Cruchley AT. Immunocytochemical examination of immune cells in periapical granulomata and odontogenic cysts. J Oral Pathol 1988;17:84 –90. 10. Akhlaghi E, Dourov N. Langerhans cells in odontogenic cysts: a retrospective study based on 142 cases. Bull Group Int Rech Sci Stomatol Odontol 1995;38:71– 6. 11. Murase N, Tatemoto Y, Iwai Y, Okada Y, Mori M. Langerhans cells in odontogenic tumours and cysts as detected by S-100 protein immunohistochemistry. Basic Appl Histochem 1990;34:135– 41.