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Ultrastructural changes in Langerhans cells in gingival overgrowth in cyclosporin A-treated renal transplant patients
Pathology (June 2004) 36(3), pp. 242–246
ANATOMICAL PATHOLOGY
Ultrastructural changes in Langerhans cells in gingival overgrowth in cyclosporin A-treated renal transplant patients GIUSTINIANO MARIANI*, CARLA CALASTRINI{, ARRIGO ALEOTTI{, EDVIGE CARNEVALE* GIORDANO STABELLINI§
AND
*Department of Morphology and Embryology, Section of Anatomy, {Department of Morphology and Embryology, Section of Histology and Embryology and {Center of Electron Microscopy, University of Ferrara; §Department of Human Morphology-Lita Segrate, University of Milano, Italy
Summary Aim: Our research was focused on the ultrastructural features of gingival epithelium in kidney transplant patients and, in particular, on Langerhans cells, with the aim of verifying whether ultrastructural modifications might explain gingival overgrowth. Methods: Using electron microscopy and immunohistochemical S100 staining at optical microscopy, we examined gingival samples obtained from 18 kidney transplant patients who presented with gingival overgrowth following cyclosporin A treatment. Results: The hyperkeratosis shown by the epithelium, and especially the absence of Birbeck granules in the Langerhans cells observed in serial sections, lead us to correlate these data to an immunodeficiency which affects the epithelium in the complex mechanism determining overgrowth. Conclusions: In our previous studies we attributed the responsibility of overgrowth to the connective tissue alone. However, in the light of the present results, we cannot exclude a contribution of the epithelium to gingival overgrowth. Key words: Langerhans cells, cyclosporin A, gingival overgrowth, gingival epithelium. Received 16 June, revised 18 November 2003, accepted 14 January 2004
INTRODUCTION In clinical practice, the immunosuppressive efficacy of cyclosporin A (CyA) is widely known, and its use has revolutionised therapeutic possibilities in the organ transplant field in just a few years. Indeed, thanks to its remarkable ability to prevent rejection, the mortality of transplant patients has been considerably reduced, and the survival of transplanted organs has increased significantly. CyA is widely used also in the treatment of different autoimmune diseases, such as insulin-dependent diabetes mellitus,1 Behcet’s disease2 and systemic lupus erythematosus,3 although it is not without side effects, including nephrotoxicity,4 hepatotoxicity,5 neurotoxicity,6 lymphoproliferative neoplasms7 and gingival overgrowth.8 The Langerhans cells (LC), first described by Paul Langerhans in 18689 as clear cells in the suprabasal layers of human epidermis, were later identified in some stratified
squamous epithelia as oral mucosa,10 vaginal11 and oesophageal mucosa.12 They are also present in the dermis, in lymph nodes and in lymphatic vessels crossing the skin, and also in the thymus, although the largest number are found in the epidermis, where they represent 2–4% of the total cell population.13 Numerous studies14–16 have dealt with their origins, properties and functions. Other authors17 have unequivocably demonstrated that LC come from a pool of precursor cells originating from bone marrow, the CD34-positive stem cells, probably influenced by granulocyte-macrophage colony-stimulating factor (GM-CSE), interleukin (IL)-3, tumour necrosis factor (TNF)-a and transforming growth factor (TGF)-b.18–20 It is now generally acknowledged that LC represent the most peripheral outpost of the immune system, and that they function as a critical link between the extracutaneous environment and the organism,21 by virtue of their capacity to detect and process antigenic molecules and present them to T lymphocytes, thus setting off a sequence of cell responses which induce immunosuppressive efficacy. According to some authors, the low density of LC in invasive squamous cell carcinoma compared with the normal state may reflect poor local immunosurveillance, and may be related to progression and spread of the tumour.22 Occurrence of malignancies in immunosuppressed organ transplant recipients is frequent.23 Wart and skin tumours are common in renal transplant recipients: immunosuppressive therapy plays an important role in predisposing patients to cutaneous malignancy.24 Other authors25 suggest that inhibition of contact allergic skin reactions by CyA may be due in part to an impairment of the function of LC. Moreover, recent studies have examined the importance of other factors for in vitro LC development, such as the role of TGF-b, and the comparison of effects of TGF-b and CyA on LC.26,27 Our previous research28–30 on the ultrastructural and histochemical features of kidney transplant patients’ gingiva, showed overgrowth as a side effect following CyA treatment, highlighting in particular the morphological aspect of the gingival lamina propria and the modifications arising in ground substance. The present study, however, has specifically addressed the ultrastructural features of the epithelium of the same gingiva and the cells it is formed by, with the particular aim of gathering information about the presence, distribution and
ISSN 0031-3025 printed/ISSN 1465–3931 # 2004 Royal College of Pathologists of Australasia DOI: 10.1080/00313020410001692585
LANGERHANS CELLS IN CYCLOSPORIN A GINGIVAL OVERGROWTH
characteristics of Langerhans cells, and in order to verify whether there are any ultrastructural modifications in the epithelium which might explain gingival overgrowth.
MATERIALS AND METHODS Ultrastructural examination was carried out on attached gingival biopsies obtained from 18 male kidney transplant patients, ranging between 30 and 60 years old, who were undergoing treatment at the Dental Clinic of the University of Ferrara, Italy, because they showed CyA-induced gingival overgrowth. All patients received i.v. administration of CyA for 2 weeks after the transplant at a dose of 10 mg/kg per day, and then a dose of 5 or 6 mg/kg per day, as well as methylprednisolone (20 mg/day, gradually reduced to a maintenance dose of 5 mg/day). Gingival overgrowth appeared in all patients about 3 months following treatment. Biopsies of attached gingiva were taken only when the plaque index was lower than 0.7. The local anaesthetic was administered by infiltration into mucosa of vestibularis fornix, but not directly into attached gingiva selected for biopsy. The samples were fixed in glutaraldehyde (2.5% in 0.1 M phosphate buffer) for 4 h at 4‡C, post-fixed in osmium tetroxide (2% in 0.1 M phosphate buffer) for 1 h at 4‡C, and then embedded in araldite ACM (Fluka, Switzerland). Serial ultrathin sections were stained with uranyl acetate and lead citrate, and examined by a Hitachi H-800 (Hitachi, Japan) transmission electron microscope (TEM) at an accelerating voltage of 100 kV. Controls consisted of uninflamed gingival biopsies from healthy patients (of similar age and gender), not taking medication and following accurate oral hygiene. For light microscopy, the same samples were fixed in 10% formalin, routinely processed and embedded in paraffin. 4-mm-thick sections were stained with H&E. LC were shown using standard techniques with antibodies against S100 protein.24,31 Quantitative analysis was performed at 6400 magnification, using a grid in the eyepiece of a Leitz Orthoplan microscope (Leitz, Germany) measuring an area of 0.29 mm2. The LC were counted (five fields per slide) in ten transverse 5-mm-thick serial sections cut every 15 mm. Statistical analysis
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assumed a ‘squamous’ appearance, had an extremely dense cytoplasm, without nucleus or organelles, which were almost completely replaced by bundles of microfilaments. The cell membrane appeared greatly thickened. Very occasionally it was possible to detect the presence of desmosomes between adjacent cells; however, they were small and presented ultrastructural modifications mainly consisting of a lack of microfilaments binding to them. The most external part of the horny layer presented desquamation, while the underlying part formed a compact stratum (Fig. 1). On the whole, the surface epithelium showed a high degree of keratinisation. Keratinocytes are the predominant type of cell in gingival epithelium. Nevertheless, it is possibile to encounter cells with different structural features. These cells include melanocytes, Merkel cells and Langerhans cells. All of these cells, except Merkel cells, are without desmosomes. Melanocytes Under optical microscope (OM), melanocytes were observed as cells with optically empty cytoplasm and thin cytoplasmic prolongations, which extended from the cell bodies and entered the spaces between the adjacent keratinocytes, taking on the appearance of dendritic cells. Under TEM, due to the lack of intercellular junctions, the melanocyte was seen to be free of connections with the adjacent keratinocytes, sending out thin cell extensions between them. Its nucleus was rounded, with a regular outline. The cytoplasm displayed wrinkled endoplasmic reticulum, highly developed Golgi apparatus, and above all, dense, oval organules delimited by membrane (Fig. 2). Merkel cells Rare cells, hard to distinguish by OM, were present in the basal layer of the epithelium. Unlike the melanocyte and the LC, the Merkel cell was not dendritic, and it had some tonofilaments and occasionally desmosomes which bound
All values are expressed as mean¡SD of the independent biopsies, each performed in duplicate. The statistical analysis was made using the Student’s t-test for paired and unpaired data. P valuesƒ0.05 were considered significant.
RESULTS The squamous stratified epithelium of gingiva in kidney transplant patients following CyA treatment showed cells disposed in four layers: basal, spinous, granular and horny. Under TEM, the basal cells presented a clearly evident nucleus and cytoplasm rich in bundles of microfilaments. The basal lamina was reinforced by a layer of connective microfibrils. The plasmalemma of the basal surface presented hemidesmosomic junctions. The spinous layer consisted of several strata of cells connected to one another by thin prolongations which established desmosomic junctions between adjacent cells, conferring the overall ‘spinous’ appearance. The granular layer was made up of cells which presented intensely osmophilic granules in their cytoplasm. The horny or cornified layer was characterised by several flattened cells arranged in numerous superimposed lamellae, thus determining its thickness. These cells, which
Fig. 1 Epithelium of attached gingiva in patients with CyA-induced gingival overgrowth. The cells of the horny layer, extremely flattened and without nuclei, present cytoplasm filled with microfilament bundles. Some desmosomes can be observed between adjacent cells. The cell membrane appears greatly thickened. F, microfilaments; D, desmosome (uranyl acetate and lead citrate staining, original magnification, 621 000).
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Fig. 2 Epithelium of attached gingiva in patients with CyA-induced gingival overgrowth. Melanocyte, with cytoplasm rich in pigment granules, between keratinocytes. M, melanocyte; BC, basal cell (uranyl acetate and lead citrate staining, original magnification, 66000).
Pathology (2004), 36(3), June
Fig. 5 Epithelium of attached gingiva in patients with CyA-induced gingival overgrowth. Scarce Langerhans cells, evident keratinisation. K, keratine lamellae; L, Langerhans cells (S100 protein immunostaining, original magnification, 6100).
osmophilic granules and small vesicles surrounded by membrane, sometimes situated near a nerve fibre associated with the cell.
Fig. 3 Epithelium of attached gingiva in patients with CyA-induced gingival overgrowth. Merkel cell. N, nucleus; d, desmosome; g, osmophilic granules (uranyl acetate and lead citrate staining, original magnification, 612 000).
it to the neighbouring cells. A highly specialised cell (Fig. 3); it was characterised by a very irregular, polylobate nucleus and by the existence in the cytoplasm of abundant
Langerhans cells In the samples with normal staining under OM, the LC appeared as clear cells with projections and dark nuclei. OM revealed their dendritic appearance, although it was not possible to distinguish them from melanocytes for a certainty. On the contrary, S100 immunohistochemical staining allowed us to locate and identify them easily in the suprabasal layers (Fig. 4). They appeared characteristically dark and dendritic in hypertrophic gingival epithelium, but less numerous (Fig. 5, 6) when compared with controls (130¡16 and 415¡54 per mm2, respectively). Under TEM, the LC presented a central body with thin cytoplasmic projections which extended threedimensionally also between adjacent keratinocytes, although they did not establish intercellular junctions with them. These dendritic cells, present mainly in the suprabasal layer, had an indented nucleus and displayed all aspects
Fig. 4 Epithelium of attached gingiva in control, showing numerous dark, dendritic Langerhans cells. E, epithelium; C, connective tissue; L, Langerhans cells (S100 protein immunostaining, original magnification, 6200).
LANGERHANS CELLS IN CYCLOSPORIN A GINGIVAL OVERGROWTH
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Fig. 6 Number of Langerhans cells per mm2 of epithelium. The values are the mean¡SD of 18 independent biopsies. CyA, CyA-treated gingival overgrowth. *Pƒ0.01 as compared with controls. Fig. 8 Epithelium of attached gingiva in control tissue. Between the cells of spinous layer the section reveals a Langerhans cell: cytoplasm presents mitochondria, cisternae of rough endoplasmic reticulum and Birbeck granules. B, Birbeck granules; M, mitochondrion (uranyl acetate and lead citrate staining, original magnification, 618 000).
Fig. 7 Epithelium of attached gingiva in patients with CyA-induced gingival overgrowth. Section of Langerhans cell which, despite ultrastructural characteristics typical of this cell (dendritic appearance, indented nucleus, absence of desmosome), does not present Birbeck granules. N, nucleus; M, mitochondrion; G, Golgi apparatus (uranyl acetate and lead citrate staining, original magnification, 615 000).
typical of metabolically active cells: highly developed Golgi apparatus, evident endoplasmic reticulum, numerous mitochondria, centrioles and lysosomal structures (Fig. 7). Nevertheless, in our observations of the LC cytoplasm in serial sections, we did not encounter Birbeck granules (BG), which constitute a distinguishing feature found exclusively in this type of cell, although they were always present in the controls (Fig. 8, 9). On the whole, the ultrastructural features of the cells which constituted the different layers of gingival epithelium in CyA-treated kidney transplant patients were exactly superimposable with controls, especially in relation to keratinocytes, Merkel cells and melanocytes. On the other hand, our observations revealed significant differences in the LC and cells of the superficial cornified layer.
DISCUSSION The immunohistochemical analysis of our research showed a reduced number of LC, while TEM demonstrated the complete absence of specific BG, even though LC maintained, on the whole, the ultrastructural features typical of these cells. The numerous superimposed lamellae
Fig. 9 Epithelium of attached gingiva in control tissue. Enlargement of an area of Fig. 8. A cytoplasmic area with Birbeck granules (B) (uranyl acetate and lead citrate staining, original magnification, 660 000).
of the horny layer gave the epithelium characteristics of hyperkeratosis. The lack of BG in the cytoplasm of the LC present in the gingival epithelium of kidney transplant patients following CyA treatment, which was repeatedly observed in our serial sections, has strengthened the hypothesis that the BG are involved in the intricate and still little-known mechanism of the relationship between these granules and the immunological response by the LC.32,33 We are thus led to believe that CyA, in its complex action mechanism triggering immunosupression, somehow acts on LC, keeping them at a stage characterised by the absence of BG. In our opinion, the hyperkeratosis we observed is correlated to the attempt to offer mechanical, physical and chemical protection in a situation where the LC, which represent a local immunological outpost, are unable to act as a consequence of BG absence. The lack of the LC specific activity may cause localised immunodeficiency, which obstructs the epithelium in its function as immunological barrier. In conclusion, in view of the morphological results
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described here and those obtained in our previous papers,28–30 which highlighted the sole responsibility of connective tissue in gingival overgrowth, and in agreement with the observations of other authors,34 we suggest that the involvement of the epithelium in the complex mechanism determining overgrowth cannot be excluded. Moreover, we believe that the modifications undergone by LC in the epithelium of kidney transplant patients may represent localised manifestations of a wider general phenomenon which may also affect other regions. Address for correspondence: Professor G. Mariani, Dipartimento di Morfologia ed Embriologia, Sezione di Anatomia, Via Fossato di Mortara 64, 44100 Ferrara, Italy. E-mail: [email protected]
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References 1. Stiller C, Laupacis A, Dupre J, et al. Effects of cyclosporine immunosuppression in insulin-dependent diabetes mellitus of recent onset. Science 1984; 223: 1362–7. 2. French-Constant C, Wolman R, Feriant JD. Cyclosporine in Behcet’s disease. (Letter.) Lancet 1983; ii: 454–5. 3. Isenberg D, Snaith M, Al-Klader A, et al. Cyclosporin relieves arthralgia, causes angioedema. New Engl J Med 1980; 303: 754. 4. Stiller CR, Keown PA, Sinclair NR, Ulan RA. Immune responses and pharmacokinetics in human renal allograft recipient treated with cyclosporine-A. In: Calne RY, White DS, editors. International Symposium on Cyclosporine-A. New York: Elsevier, 1981. 5. Klintman GBC, Iwatsuki S, Starzl TE. Cyclosporin A hepatotoxicity in 66 renal allograft recipients. Transplantation 1981; 32: 488–9. 6. Beaman M, Parvin S, Veitch PS, Walls J. Convulsions associated with cyclosporin A in renal transplant recipients. Br Med J 1985; 290: 139. 7. Starzl TE, Porter KA, Iwatsuki S, et al. Reversability of lymphomas and lymphoproliferative lesions developing under cyclosporin-steroid therapy. Lancet 1984; i: 583–7. 8. Rostock MH, Fry HR, Turner JE. Severe gingival overgrowth associated with cyclosporin therapy. J Periodontol 1986; 57: 294–9. 9. Langerhans P. Uber die Nerven der menschlichen Haut. Virchows Arch Abt B Pathol Anat 1868; 44: 325–37. 10. Waterhouse JP, Squire CA. The Langerhans cell in human gingival epithelium. Arch Oral Biol 1967; 12: 341–8. 11. Young WG. Epithelial kinetics affect Langerhans cell of mouse vaginal epithelium. Acta Anat 1985; 123: 131–6. 12. Al Yassin T, Toner PG. Langerhans cell in the human oesophagus. J Anat 1976; 122: 435–45. 13. Stingl G, Katz SI, Clement L, Green I, Shevach EM. Immunologic functions of Ia-bearing epidermal Langerhans cells. J Immunol 1978; 121: 2005–13. 14. Ferreira-Marques J. Systema sensitivum intraepidermicum: Die Langerhanszellen als Rezeptoren des hellen Schmerzes. Doloreceptors. Arch Klin Exp Dermatol 1951; 193: 191–250. 15. Billingham RS, Medawar PB. A study of the branched cells of the mammalian epidermis with special references to the fate of their division products. Phil Trans R Soc London (Biol) 1953; 237: 151–71. 16. Breathnach AS, Silvers WK, Smith J, Heyner S. Langerhans cells in mouse skin experimentally deprived of its neural crest component. J Invest Dermatol 1968; 50: 147–60. 17. Katz SI, Tamaki K, Sachs DH. Epidermal Langerhans cells are derived from cells originating in the bone marrow. Nature 1979; 282: 324–6. 18. Caux C, Vanbervliet B, Massacrier C, et al. CD34z hematopoietic
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progenitors from human cord blood differentiate along two independent dendritic cell pathways in response to GM-CSFzTNF alpha. J Exp Med 1996; 184: 695–706. Reid CD, Stackpoole A, Meager A, Tikerpae J. Interactions of tumor necrosis factor with granulocyte-macrophage colony-stimulating factor and other cytokines in the regulation of dendritic cell growth in vitro from early bipotent CD34z progenitors in human bone marrow. J Immunol 1992; 149: 2681–8. Strobl H, Riedl E, Schneiecker C, et al. TGF-beta 1 promotes in vitro development of dendritic cells from CD34z hemopoietic progenitors. J Immunol 1996; 157: 1499–507. Stingl G, Katz SI, Green I, Shevach EM. The functional role of Langherans cells. J Invest Dermatol 1980; 74: 315–8. Tay SK, Jenkins D. Langerhans cell population in early invasive squamous cell carcinoma of the uterine cervix. Aust NZ J Obstet Gynaecol 1989; 29: 38–40. Penn I. Occurrence of malignancies in immunosuppressed organ transplant recipients. In: Abouna GM, Kumar MSA, White AG, editors. Organ Transplantation Proc 2nd East Soc Org Transplant. Dordrecht: Kluwer Academic Publisher, 1990; 475. Bedani PL, Risichella IS, Strumia R, et al. Kaposi’s sarcoma in renal transplant recipients: pathogenetic relation between the reduced density of Langerhans cells and cyclosporin A therapy. J Nephrol 1999; 12: 193–6. Teunissen MB, De Jager MH, Kapsenberg ML, BOS JD. Inhibitory effect of cyclosporin A on antigen and alloantigen presenting capacity of human epidermal Langerhans cells. Br J Dermatol 1991; 125: 309–16. Mollah ZU, Aiba S, Nakagawa S, et al. Macrophage colonystimulating factor in cooperation with transforming growth factorbetal induces the differentiation of CD34z hematopoietic progenitor cells into Langerhans cells under serum-free conditions without granulocyte-macrophage colony-stimulating factor. J Invest Dermatol 2003; 120: 256–65. Demidem A, Taylor JR, Grammer SF, Streilein JW. Comparison of effects of transforming growth factor-beta and cyclosporin A on antigen-presenting cells of blood and epidermis. J Invest Dermatol 1991; 96: 401–7. Mariani G, Calastrini C, Carinci F, Marzola R, Calura G. Ultrastructural features of Cyclosporine A-induced gingival hyperplasia. J Periodontol 1993; 64: 1092–7. Mariani G, Calastrini C, Carinci F, et al. Ultrastructural and histochemical features of the ground substance in Cyclosporin A-induced gingival overgrowth. J Periodontol 1996; 67: 21–7. Mariani G, Calastrini C, Bernabei P, Stabellini G. Aumento di volume gengivale indotto da ciclosporina. Accad Sci Ferrara, Atti 1996; 72/73: 247–67. Hachisuga T, Fukuda K, Hayashi Y, Iwasaka T, Sugimori H. Immunohistochemical demonstration of histiocytes in normal ectocervical epithelial lesions of the uterine cervix. Gynecol Oncol 1989; 33: 273–8. Hanau D, Fabre M, Schmitt DA, et al. Human epidermal Langerhans cells internalize by receptor-mediated endocytosis T6 (CD1 ‘NA1/34’) surface antigen. Birbeck granules are involved in the intercellular traffic of the T6 antigen. J Invest Dermatol 1987; 89: 172–7. Mc Dermott R, Ziylan U, Spehner D, et al. Birbeck granules are subdomains of endosomal recycling compartment in human epidermal Langerhans Cells, which form where Langerin accumulates. Mol Biol Cell 2002; 13: 317–35. Pisanty S, Rahamim E, Ben-Ezra D, Shoshan S. Prolonged systemic administration of Cyclosporin A affects gingival epitheliun. J Periodontol 1990; 61: 138–41.
ANATOMICAL PATHOLOGY
Ultrastructural changes in Langerhans cells in gingival overgrowth in cyclosporin A-treated renal transplant patients GIUSTINIANO MARIANI*, CARLA CALASTRINI{, ARRIGO ALEOTTI{, EDVIGE CARNEVALE* GIORDANO STABELLINI§
AND
*Department of Morphology and Embryology, Section of Anatomy, {Department of Morphology and Embryology, Section of Histology and Embryology and {Center of Electron Microscopy, University of Ferrara; §Department of Human Morphology-Lita Segrate, University of Milano, Italy
Summary Aim: Our research was focused on the ultrastructural features of gingival epithelium in kidney transplant patients and, in particular, on Langerhans cells, with the aim of verifying whether ultrastructural modifications might explain gingival overgrowth. Methods: Using electron microscopy and immunohistochemical S100 staining at optical microscopy, we examined gingival samples obtained from 18 kidney transplant patients who presented with gingival overgrowth following cyclosporin A treatment. Results: The hyperkeratosis shown by the epithelium, and especially the absence of Birbeck granules in the Langerhans cells observed in serial sections, lead us to correlate these data to an immunodeficiency which affects the epithelium in the complex mechanism determining overgrowth. Conclusions: In our previous studies we attributed the responsibility of overgrowth to the connective tissue alone. However, in the light of the present results, we cannot exclude a contribution of the epithelium to gingival overgrowth. Key words: Langerhans cells, cyclosporin A, gingival overgrowth, gingival epithelium. Received 16 June, revised 18 November 2003, accepted 14 January 2004
INTRODUCTION In clinical practice, the immunosuppressive efficacy of cyclosporin A (CyA) is widely known, and its use has revolutionised therapeutic possibilities in the organ transplant field in just a few years. Indeed, thanks to its remarkable ability to prevent rejection, the mortality of transplant patients has been considerably reduced, and the survival of transplanted organs has increased significantly. CyA is widely used also in the treatment of different autoimmune diseases, such as insulin-dependent diabetes mellitus,1 Behcet’s disease2 and systemic lupus erythematosus,3 although it is not without side effects, including nephrotoxicity,4 hepatotoxicity,5 neurotoxicity,6 lymphoproliferative neoplasms7 and gingival overgrowth.8 The Langerhans cells (LC), first described by Paul Langerhans in 18689 as clear cells in the suprabasal layers of human epidermis, were later identified in some stratified
squamous epithelia as oral mucosa,10 vaginal11 and oesophageal mucosa.12 They are also present in the dermis, in lymph nodes and in lymphatic vessels crossing the skin, and also in the thymus, although the largest number are found in the epidermis, where they represent 2–4% of the total cell population.13 Numerous studies14–16 have dealt with their origins, properties and functions. Other authors17 have unequivocably demonstrated that LC come from a pool of precursor cells originating from bone marrow, the CD34-positive stem cells, probably influenced by granulocyte-macrophage colony-stimulating factor (GM-CSE), interleukin (IL)-3, tumour necrosis factor (TNF)-a and transforming growth factor (TGF)-b.18–20 It is now generally acknowledged that LC represent the most peripheral outpost of the immune system, and that they function as a critical link between the extracutaneous environment and the organism,21 by virtue of their capacity to detect and process antigenic molecules and present them to T lymphocytes, thus setting off a sequence of cell responses which induce immunosuppressive efficacy. According to some authors, the low density of LC in invasive squamous cell carcinoma compared with the normal state may reflect poor local immunosurveillance, and may be related to progression and spread of the tumour.22 Occurrence of malignancies in immunosuppressed organ transplant recipients is frequent.23 Wart and skin tumours are common in renal transplant recipients: immunosuppressive therapy plays an important role in predisposing patients to cutaneous malignancy.24 Other authors25 suggest that inhibition of contact allergic skin reactions by CyA may be due in part to an impairment of the function of LC. Moreover, recent studies have examined the importance of other factors for in vitro LC development, such as the role of TGF-b, and the comparison of effects of TGF-b and CyA on LC.26,27 Our previous research28–30 on the ultrastructural and histochemical features of kidney transplant patients’ gingiva, showed overgrowth as a side effect following CyA treatment, highlighting in particular the morphological aspect of the gingival lamina propria and the modifications arising in ground substance. The present study, however, has specifically addressed the ultrastructural features of the epithelium of the same gingiva and the cells it is formed by, with the particular aim of gathering information about the presence, distribution and
ISSN 0031-3025 printed/ISSN 1465–3931 # 2004 Royal College of Pathologists of Australasia DOI: 10.1080/00313020410001692585
LANGERHANS CELLS IN CYCLOSPORIN A GINGIVAL OVERGROWTH
characteristics of Langerhans cells, and in order to verify whether there are any ultrastructural modifications in the epithelium which might explain gingival overgrowth.
MATERIALS AND METHODS Ultrastructural examination was carried out on attached gingival biopsies obtained from 18 male kidney transplant patients, ranging between 30 and 60 years old, who were undergoing treatment at the Dental Clinic of the University of Ferrara, Italy, because they showed CyA-induced gingival overgrowth. All patients received i.v. administration of CyA for 2 weeks after the transplant at a dose of 10 mg/kg per day, and then a dose of 5 or 6 mg/kg per day, as well as methylprednisolone (20 mg/day, gradually reduced to a maintenance dose of 5 mg/day). Gingival overgrowth appeared in all patients about 3 months following treatment. Biopsies of attached gingiva were taken only when the plaque index was lower than 0.7. The local anaesthetic was administered by infiltration into mucosa of vestibularis fornix, but not directly into attached gingiva selected for biopsy. The samples were fixed in glutaraldehyde (2.5% in 0.1 M phosphate buffer) for 4 h at 4‡C, post-fixed in osmium tetroxide (2% in 0.1 M phosphate buffer) for 1 h at 4‡C, and then embedded in araldite ACM (Fluka, Switzerland). Serial ultrathin sections were stained with uranyl acetate and lead citrate, and examined by a Hitachi H-800 (Hitachi, Japan) transmission electron microscope (TEM) at an accelerating voltage of 100 kV. Controls consisted of uninflamed gingival biopsies from healthy patients (of similar age and gender), not taking medication and following accurate oral hygiene. For light microscopy, the same samples were fixed in 10% formalin, routinely processed and embedded in paraffin. 4-mm-thick sections were stained with H&E. LC were shown using standard techniques with antibodies against S100 protein.24,31 Quantitative analysis was performed at 6400 magnification, using a grid in the eyepiece of a Leitz Orthoplan microscope (Leitz, Germany) measuring an area of 0.29 mm2. The LC were counted (five fields per slide) in ten transverse 5-mm-thick serial sections cut every 15 mm. Statistical analysis
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assumed a ‘squamous’ appearance, had an extremely dense cytoplasm, without nucleus or organelles, which were almost completely replaced by bundles of microfilaments. The cell membrane appeared greatly thickened. Very occasionally it was possible to detect the presence of desmosomes between adjacent cells; however, they were small and presented ultrastructural modifications mainly consisting of a lack of microfilaments binding to them. The most external part of the horny layer presented desquamation, while the underlying part formed a compact stratum (Fig. 1). On the whole, the surface epithelium showed a high degree of keratinisation. Keratinocytes are the predominant type of cell in gingival epithelium. Nevertheless, it is possibile to encounter cells with different structural features. These cells include melanocytes, Merkel cells and Langerhans cells. All of these cells, except Merkel cells, are without desmosomes. Melanocytes Under optical microscope (OM), melanocytes were observed as cells with optically empty cytoplasm and thin cytoplasmic prolongations, which extended from the cell bodies and entered the spaces between the adjacent keratinocytes, taking on the appearance of dendritic cells. Under TEM, due to the lack of intercellular junctions, the melanocyte was seen to be free of connections with the adjacent keratinocytes, sending out thin cell extensions between them. Its nucleus was rounded, with a regular outline. The cytoplasm displayed wrinkled endoplasmic reticulum, highly developed Golgi apparatus, and above all, dense, oval organules delimited by membrane (Fig. 2). Merkel cells Rare cells, hard to distinguish by OM, were present in the basal layer of the epithelium. Unlike the melanocyte and the LC, the Merkel cell was not dendritic, and it had some tonofilaments and occasionally desmosomes which bound
All values are expressed as mean¡SD of the independent biopsies, each performed in duplicate. The statistical analysis was made using the Student’s t-test for paired and unpaired data. P valuesƒ0.05 were considered significant.
RESULTS The squamous stratified epithelium of gingiva in kidney transplant patients following CyA treatment showed cells disposed in four layers: basal, spinous, granular and horny. Under TEM, the basal cells presented a clearly evident nucleus and cytoplasm rich in bundles of microfilaments. The basal lamina was reinforced by a layer of connective microfibrils. The plasmalemma of the basal surface presented hemidesmosomic junctions. The spinous layer consisted of several strata of cells connected to one another by thin prolongations which established desmosomic junctions between adjacent cells, conferring the overall ‘spinous’ appearance. The granular layer was made up of cells which presented intensely osmophilic granules in their cytoplasm. The horny or cornified layer was characterised by several flattened cells arranged in numerous superimposed lamellae, thus determining its thickness. These cells, which
Fig. 1 Epithelium of attached gingiva in patients with CyA-induced gingival overgrowth. The cells of the horny layer, extremely flattened and without nuclei, present cytoplasm filled with microfilament bundles. Some desmosomes can be observed between adjacent cells. The cell membrane appears greatly thickened. F, microfilaments; D, desmosome (uranyl acetate and lead citrate staining, original magnification, 621 000).
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Fig. 2 Epithelium of attached gingiva in patients with CyA-induced gingival overgrowth. Melanocyte, with cytoplasm rich in pigment granules, between keratinocytes. M, melanocyte; BC, basal cell (uranyl acetate and lead citrate staining, original magnification, 66000).
Pathology (2004), 36(3), June
Fig. 5 Epithelium of attached gingiva in patients with CyA-induced gingival overgrowth. Scarce Langerhans cells, evident keratinisation. K, keratine lamellae; L, Langerhans cells (S100 protein immunostaining, original magnification, 6100).
osmophilic granules and small vesicles surrounded by membrane, sometimes situated near a nerve fibre associated with the cell.
Fig. 3 Epithelium of attached gingiva in patients with CyA-induced gingival overgrowth. Merkel cell. N, nucleus; d, desmosome; g, osmophilic granules (uranyl acetate and lead citrate staining, original magnification, 612 000).
it to the neighbouring cells. A highly specialised cell (Fig. 3); it was characterised by a very irregular, polylobate nucleus and by the existence in the cytoplasm of abundant
Langerhans cells In the samples with normal staining under OM, the LC appeared as clear cells with projections and dark nuclei. OM revealed their dendritic appearance, although it was not possible to distinguish them from melanocytes for a certainty. On the contrary, S100 immunohistochemical staining allowed us to locate and identify them easily in the suprabasal layers (Fig. 4). They appeared characteristically dark and dendritic in hypertrophic gingival epithelium, but less numerous (Fig. 5, 6) when compared with controls (130¡16 and 415¡54 per mm2, respectively). Under TEM, the LC presented a central body with thin cytoplasmic projections which extended threedimensionally also between adjacent keratinocytes, although they did not establish intercellular junctions with them. These dendritic cells, present mainly in the suprabasal layer, had an indented nucleus and displayed all aspects
Fig. 4 Epithelium of attached gingiva in control, showing numerous dark, dendritic Langerhans cells. E, epithelium; C, connective tissue; L, Langerhans cells (S100 protein immunostaining, original magnification, 6200).
LANGERHANS CELLS IN CYCLOSPORIN A GINGIVAL OVERGROWTH
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Fig. 6 Number of Langerhans cells per mm2 of epithelium. The values are the mean¡SD of 18 independent biopsies. CyA, CyA-treated gingival overgrowth. *Pƒ0.01 as compared with controls. Fig. 8 Epithelium of attached gingiva in control tissue. Between the cells of spinous layer the section reveals a Langerhans cell: cytoplasm presents mitochondria, cisternae of rough endoplasmic reticulum and Birbeck granules. B, Birbeck granules; M, mitochondrion (uranyl acetate and lead citrate staining, original magnification, 618 000).
Fig. 7 Epithelium of attached gingiva in patients with CyA-induced gingival overgrowth. Section of Langerhans cell which, despite ultrastructural characteristics typical of this cell (dendritic appearance, indented nucleus, absence of desmosome), does not present Birbeck granules. N, nucleus; M, mitochondrion; G, Golgi apparatus (uranyl acetate and lead citrate staining, original magnification, 615 000).
typical of metabolically active cells: highly developed Golgi apparatus, evident endoplasmic reticulum, numerous mitochondria, centrioles and lysosomal structures (Fig. 7). Nevertheless, in our observations of the LC cytoplasm in serial sections, we did not encounter Birbeck granules (BG), which constitute a distinguishing feature found exclusively in this type of cell, although they were always present in the controls (Fig. 8, 9). On the whole, the ultrastructural features of the cells which constituted the different layers of gingival epithelium in CyA-treated kidney transplant patients were exactly superimposable with controls, especially in relation to keratinocytes, Merkel cells and melanocytes. On the other hand, our observations revealed significant differences in the LC and cells of the superficial cornified layer.
DISCUSSION The immunohistochemical analysis of our research showed a reduced number of LC, while TEM demonstrated the complete absence of specific BG, even though LC maintained, on the whole, the ultrastructural features typical of these cells. The numerous superimposed lamellae
Fig. 9 Epithelium of attached gingiva in control tissue. Enlargement of an area of Fig. 8. A cytoplasmic area with Birbeck granules (B) (uranyl acetate and lead citrate staining, original magnification, 660 000).
of the horny layer gave the epithelium characteristics of hyperkeratosis. The lack of BG in the cytoplasm of the LC present in the gingival epithelium of kidney transplant patients following CyA treatment, which was repeatedly observed in our serial sections, has strengthened the hypothesis that the BG are involved in the intricate and still little-known mechanism of the relationship between these granules and the immunological response by the LC.32,33 We are thus led to believe that CyA, in its complex action mechanism triggering immunosupression, somehow acts on LC, keeping them at a stage characterised by the absence of BG. In our opinion, the hyperkeratosis we observed is correlated to the attempt to offer mechanical, physical and chemical protection in a situation where the LC, which represent a local immunological outpost, are unable to act as a consequence of BG absence. The lack of the LC specific activity may cause localised immunodeficiency, which obstructs the epithelium in its function as immunological barrier. In conclusion, in view of the morphological results
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described here and those obtained in our previous papers,28–30 which highlighted the sole responsibility of connective tissue in gingival overgrowth, and in agreement with the observations of other authors,34 we suggest that the involvement of the epithelium in the complex mechanism determining overgrowth cannot be excluded. Moreover, we believe that the modifications undergone by LC in the epithelium of kidney transplant patients may represent localised manifestations of a wider general phenomenon which may also affect other regions. Address for correspondence: Professor G. Mariani, Dipartimento di Morfologia ed Embriologia, Sezione di Anatomia, Via Fossato di Mortara 64, 44100 Ferrara, Italy. E-mail: [email protected]
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