Identity of TUNEL-positive cells in the oral buccal epithelium of normal mucosa and lichen lesions

BACKGROUND: In situ detection of DNA fragmentation by TdT (terminal deoxynucleotidyl transferase)-mediated dUTP nick end labeling (TUNEL) is a widely used technique to identify apoptotic cells in the terminal phases of cell death. Several studies have shown that there are statistically increased numbers of TUNEL(+) cells within the epithelium of oral lichen (OL). It was suggested that this indicates an increased rate of apoptosis among basal and suprabasal keratinocytes in OL epithelium. The aim of this study was to identify the TUNEL(+) cells in the epithelium of erythematous (ERY) OL and normal oral mucosa (NOM). METHODS: Sections of biopsies from NOM and ERY OL were processed for TUNEL combined with immunostaining for pan-cytokeratin or for cell markers specifically expressed by different leukocytes. RESULTS: In NOM, TUNEL(+) keratinocytes were almost exclusively seen in the outermost epithelial layers. This labeling was absent in ERY OL. In the basal and lower spinous layers, more TUNEL(+) cell nuclei were seen in ERY OL as compared with NOM, in accordance with previous studies. The present observations show, however, that only very few of these cells were keratinocytes, but rather were CD4(+) lymphocytes and CD68(+) macrophages. There was no difference between the numbers of TUNEL(+) keratinocytes in basal and lower spinous layers in ERY OL and NOM epithelium. No intraepithelial CD8(+) lymphocytes, Langerhans cells, or mast cells were found to be TUNEL(+). CONCLUSION: The findings indicate that the pathologic changes in ERY OL epithelium cannot be explained by increased prevalence of terminal keratinocyte cell death identified by TUNEL.     

Interepithelial cells of the oral mucosa Light and electron microscopic observations in germfree, specific pathogen-free and conventionalized mice

Abstract Interepithelial cells are found in all epithelia of the internal and external surfaces of the mammalian body. The regional differences of these Interepithelial cells and their function are not completely known so far. The quantitative and qualitative changes of the interepithelial cell population were investigated in germfree, specific pathogen-free and conventionalized mice by light and electron microscopy. Germfree and specific pathogen-free animals did not show significant differences in the number of interepithelial cells. In the epithelium of the tongue a mean of 7.4 cells per 1000 basal cells is found. After conventionalization a significant increase to 14.4 interepithelial cells per 1000 basal cells is observed. The number of cells in the buccal epithelium is constantly about 20% higher than in the epithelium of the tongue. In the oral mucosa lymphocytes, cerebriform cells and Langerhans cells are an integral component of the epithelium. In contrast to the monostratified intestinal mucosal epithelium, which is considered a secondary lymphatic tissue, the interepithelial lymphocytes of the oral mucosa are not significantly decreased in germfree animals. This could indicate that the oral mucosa functions partly as a primary lymphatic tissue. Interepithelial cerebriform cells and Langerhans cells increased after conventionalization with a maximum after 10 days in response to exogenous antigens. Both cells are immunologically important. The observations prove that the oral mucosa represents a local immunologic system in which the Langerhans cell plays an important part by formation a reticulo-epithelial tissue.     

Human gingival Langerhans cells in health and disease

Epithelial Langerhans cells in samples of healthy and diseased gingival tissue were studied using ATPase histochemistry and the monoclonal antibodies OKT6 and anti HLA-DR. In healthy gingiva Langerhans cells were seen in both oral and sulcular epithelium; they were generally positioned in the basal layers. No Langerhans cells were seen in junctional epithelium. In diseased tissue there was a large increase in the number of Langerhans cells in both oral and sulcular epithelium with many more being situated in the stratum spinosum. There was an increase in the expression of the Class 2 antigen, HLA-DR, and morphological polarization occurred with dendrites preferentially orientated towards the surface. No Langerhans cells were seen in the pocket lining epithelium of periodontally diseased gingiva.     

Pattern of distribution of T lymphocytes, Langerhans cells and HLA-DR bearing cells in normal human oral mucosa

The tissue distribution of helper/inducer and suppressor/cytotoxic T cells, Langerhans cells (LC) and HLA-DR bearing cells was determined in normal oral mucosa by use of monoclonal antibodies OKT4, OKT8, OK.T6 and OKIal, respectively. OKT4+ and OKT8+ cells were invariably present in normal oral epithelium and in the lamina propria. OKT8+ cells were consistently seen inside the basal cell layer of the epithelium. The distribution of LC in oral epithelium showed regional variation. In palatal epithelium LC were evenly distributed in the basal half of the epithelium, whereas in buccal mucosa the highest concentration of LC was seen in the epithelium overlying the tips of connective tissue papillae. OKIal stained dendritic cells in the epithelium and plump cells with small dendritic processes in the connective tissue. Some of the latter were located close to the basal cells of the epithelium. The consistent relationship between immunocompetent cells and the epithelium of the oral mucosa suggests the presence of a local immunologic defence barrier in the oral mucosa.     

Pattern of distribution of T lymphocytes, Langerhans cells and HLA-DR bearing cells in normal human oral mucosa

The tissue distribution of helper/inducer and suppressor/cytotoxic T cells, Langerhans cells (LC) and HLA-DR bearing cells was determined in normal oral mucosa by use of monoclonal antibodies OKT4, OKT8, OKT6 and OKIa1, respectively. OKT4+ and OKT8+ cells were invariably present in normal oral epithelium and in the lamina propria. OKT8+ cells were consistently seen inside the basal cell layer of the epithelium. The distribution of LC in oral epithelium showed regional variation. In palatal epithelium LC were evenly distributed in the basal half of the epithelium, whereas in buccal mucosa the highest concentration of LC was seen in the epithelium overlying the tips of connective tissue papillae. OKIa1 stained dendritic cells in the epithelium and plump cells with small dendritic processes in the connective tissue. Some of the latter were located close to the basal cells of the epithelium. The consistent relationship between immunocompetent cells and the epithelium of the oral mucosa suggests the presence of a local immunologic defence barrier in the oral mucosa.     

Frequency and distribution of binucleate cells in oral epithelium of several species of laboratory rodents

Keratinocytes in certain regions of rodent oral epithelium have a tendency to form a peculiar type of binucleate cell. The nuclei, in contrast to those in binucleate cells in most other tissues, are closely apposed with a flat interface, tetraploid and of approx. equal size. Binucleate cells were absent from the oral epithelium of several unrelated non-rodent mammalian species, but they occurred in descending order of frequency in guinea pig, rat, hamster and mouse; they were in epithelia of the lining mucosa, but absent from epithelia of the masticatory mucosa. In buccal epithelium of the rat, they constituted 2.5 per cent of the basal cells and 8.2 per cent of the lower spinous cells. This frequency was maintained in the succeeding cell layers. Regions of stimulated epithelium in zinc-deficient rats showed a four-fold increase over controls in frequency in the germinative cell layers and persistently higher frequency in succeeding layers, including in parakeratin. The mode of formation of the cells is unknown, but it was not associated with a lag in cytoplasmic division.     

Ultrastructural observation on Langerhans cells in the rat gingival epithelium

The present study was carried out to investigate ultrastructurally Langerhans cells in the rat gingival epithelium. The gingivae of lower incisors of 15 Wistar rats were examined by electron microscopy. The results were as follows: Langerhans cells were observed mainly in the lower prickle-cell layer of the gingival epithelium. On rare occasions Langerhans cells were also found in the basal and granular layers. The average number of Langerhans cells per 100 cells in the prickle-cell layer was 1.0 cell. Usually Langerhans cells had clear cytoplasms and convoluted or indented nuclei, although sometimes the cells exhibited round nuclei. The clear cytoplasm contained a moderately developed Golgi apparatus and a small number of rough surfaced endoplasmic reticulum, free ribosomes, mitochondria, and lysosomes, but it lacked tonofilaments. Birbeck granules were often found in close vicinity of the Golgi apparatus. The average number of Birbeck granules per one Langerhans cell was 4.3 granules. The cell membrane of Langerhans cells had no junctional complexes like desmosomes. The degeneration of keratinocytes adjacent to Langerhans cells was observed in a few specimens.     

Identification of inflammatory cell phenotypes in human oral carcinomas by means of monoclonal antibodies

Monoclonal antibodies reacting with human T cell sub-populations, Langerhans cells and macrophages were used to examine the quantitative distribution of immune-competent cells in normal oral mucosa and invasive oral carcinomas. Both immunofluorescent and immunoperoxidase procedures were applied. In normal oral epithelia, the dominant immune-reactive cell was the Langerhans cell, positive for OKT 6 and expressing HLA-DR gene products (OKIal⁺). Many intra-epithelial non-epithelial cells (non-keratinocytes), belonged to the lymphocyte system carrying the suppressor/cytotoxic phenotype (OKT 8⁺). This lymphocyte sub-population was also the most prominent cell type in the normal mucosal stroma. The quantitative evaluation of immune-competent cells in squamous cell carcinomas revealed elevated numbers of all the inflammatory cell sub-populations investigated (suppressor/cytotoxic lymphocytes, helper/inducer lymphocytes, Langerhans cells, macrophages) compared with the normal oral mucosa. There was a striking increase in suppressor/cytotoxic lymphocytes (OKT 8⁺) and in cells of the macrophage system, including Langerhans cells (OKIal⁺, OKM l⁺, OKT 6⁺). In the stroma distant to the tumour complexes, many helper/inducer lymphocytes (OKT 4⁺) were also observed.     

The ultrastructure of human gingival Langerhans cells in health and disease

There was a statistically significant shift towards increased proportions of type I Langerhans cells (containing many Langerhans-cell granules) and reduced proportions of both type II Langerhans cells (containing few granules) and indeterminate cells in diseased oral epithelium when compared to healthy oral epithelium. Langerhans cells and indeterminate cells were also seen in the sulcular epithelium of healthy and diseased specimens but never in junctional or pocket-lining epithelium.     

Hertwigs sheath in the rat incisor

The histodynamic nature of Hertwig's sheath of the mandibular rat incisor was studied utilizing tritiated thymidine. The epithelial sheath is composed of three distinct cell layers: (1) inner dental epithelium, (2) stellate reticulum, and (3) outer dental epithelium. Incisal to the basal loop area, the outer layers eventually diminish and disappear leaving only the inner dental epithelium. This layer ultimately becomes perforated and is replaced by connective tissue elements. Epithelial rest cells are not identifiable in the periodontal ligament. Autoradiographic study shows labeling to be more frequent in the inner dental epithelium than the other two epithelial layers. At the one hour period, 97 per cent of the labeled cells in the inner dental epithelium are located in the basal two-thirds of the area between the basal loop and the site of predentin deposition. A study of cell kinetics indicates that the inner dental epithelium is made up of a renewing cell population. The cells have a total generation time of 25.3 hours; a synthesis period of nine hours; a G₂ period of two to six hours; a mitotic period of approximately one-half hour, and a G₁ period ranging from 9.8 to 13.8 hours. The rate of cell migration is of the same order as the rate of tooth eruption (358 μ per day).     

Cellular sources of transforming growth factor-alpha in human oral cancer

Aberrant expression of TGF-alpha is associated with human malignant oral epithelium. Experiments were initiated to determine the cellular sources of transforming growth factor-alpha (TGF-alpha) in human oral cancer. Ten freshly resected human oral cancers and four specimens of normal human oral epithelium were studied by in situ hybridization and immunohistochemistry. Tissues were probed with 35S-labeled sense and antisense riboprobes to (i) human TGF-alpha (hTGF-alpha), (ii) human epidermal growth factor receptor (EGFR) to determine the distribution of TGF-alpha responsive cells, and (iii) histone H3 to examine TGF-alpha and/or EGFR's possible contribution to altered proliferation in transformed epithelium. Results of our experiments showed that TGF-alpha mRNA could be detected in normal and transformed human oral epithelium. More surprising, we have identified the major source of TGF-alpha mRNA to be the infiltrating eosinophils. A monoclonal antibody to the mature human TGF-alpha peptide stained similar areas in normal and malignant specimens. Eosinophils associated with tumors exhibited positive cytoplasmic immunostaining for TGF-alpha protein. Labeling of EGFR mRNA in human oral epithelium demonstrated uniform labeling of basal layers in normal, hyperplastic, and mildly dysplastic epithelium. In severely dysplastic epithelium and carcinomas (particularly moderate to poorly differentiated types), cellular levels of EGFR mRNA were significantly higher. The profile of altered cellular levels of EGFR mRNA correlated well with the profile of altered proliferation as indicated by H3 mRNA labeling. We hypothesize that the overproduction of EGFR mRNA in tumor epithelium--together with the localized delivery of high amounts of TGF-alpha by eosinophils at tumor-developing sites--is responsible for the increased proliferation of the tumor epithelium.     

Peculiarities of protein Ki-67 expression in cases of leukoplakia and epidermoid cancer of oral mucous membrane

There were presented results of the study directed to disclosure of malignant cell changes criterion in cases of oral mucous membrane leukoplakia with different degrees of neoplastic transformation according to WHO-2005 classification of squamous intraepithelial neoplasia (SIN). With the help of immunohistochemical method proliferation in different layers of oral mucous membrane epithelium was evaluated. It was established that most important for diagnostics was the correspondence of proliferating cells in parabasal and basal epithelium layers. Figure less than 1 was corresponding to normal epithelium and SIN1, between 1 and 2 was corresponding to SIN2, 2 and more was characteristic itoSIN3.     

Quantitative assessment of apoptosis in oral lichen planus.

OBJECTIVE: The aims of this study were to examine the frequency of apoptoses in oral lichen planus by in situ end labeling, to ascertain whether this technique is as sensitive as conventional histologic analysis, and to examine the effect of lymphocytic infiltration. STUDY DESIGN: Numbers of apoptoses in hematoxylin-eosin stained sections were compared with numbers of apoptotic nuclei identified by in situ end labeling in oral lichen planus (n = 26) and normal buccal epithelium (n = 8). Immunohistochemical staining with MIB-1 and for Bcl-2 and Bax enabled possible regulatory pathways to be investigated. RESULTS: In oral lichen planus, approximately 1 apoptotic cell was detected per millimeter of basal layer, cell death increasing with lymphocytic infiltration. Epithelial cell proliferation did not correlate with apoptosis. Bcl-2 expression was weak or absent in basal cells, and Bax was localized to upper prickle cells. CONCLUSIONS: Increased numbers of apoptoses were detected in oral lichen planus, especially in association with lymphocytic infiltration, higher numbers being seen with hematoxylin-eosin staining than with in situ end labeling.     

Quantitative analysis of immunocompetent cells in human normal oral and uterine cervical mucosa, oral papillomas and leukoplakias

Using monoclonal antibodies reacting with T-cell subpopulations, Langerhans cells and macrophages, the number and distribution of cells of the immune system in normal oral and cervical mucosa was determined and statistically compared with that in oral papillomas and oral leukoplakias. Increased numbers of labelled cells were found in oral leukoplakias and particularly in oral papillomas. In the epithelium of all specimens, Langerhans cells and T-lymphocytes of the suppressor/cytotoxic phenotype as well as of the helper phenotype were seen. Suppressor/cytotoxic and helper T-lymphocytes were in equal numbers in the epithelium of oral papillomas, but were about 2:1 in all other lesions. In normal oral epithelium, macrophages were rare but were in greater numbers in leukoplakias and papillomas. In the connective tissue of all lesions, more labelled cells were present than in epithelium with T-lymphocytes predominant. Although Langerhans cells were rare in connective tissue, many were seen in oral papillomas.     

Association between plaque accumulation and Langerhans cell numbers in the oral epithelium of attached gingiva

Abstract After gingival health had been achieved in four subjects they were instructed to cease all oral hygiene measures. At 0, 8 and 21 days Plaque and Gingival Indices were recorded and gingival biopsies were removed from the buccal aspect of a first molar. Frozen sections of the gingival oral epithelium were stained for ATPase and 5′-nucleotidase to determine the number of Langerhans cells in a defined cross-sectional area. It was found that, as plaque accumulated, there was a statistically significant increase in the number of Langerhans cells in oral epithelium, particularly in the stratum spinosum. These results indicate that dental plaque can elicit a response in Langerhans cells located in the oral epithelium of the gingiva.     

Extent and diversity of inflammatory cell infiltrates in squamous cell carcinomas and basal cell epitheliomas of the head and neck

Using monoclonal antibodies reactive with Langerhans' cells (LCs), macrophages, and T cell subpopulations, the density and proportions of cells of the immune system of the normal oral mucosa were determined immunohistochemically, and compared with findings in oral squamous cell carcinomas (SCC) and basal cell epitheliomas (BCE). In normal oral epithelia, the dominant cell type was the LC, positive for CD 1, and expressing HLA-DR antigens (DR+). Many intraepithelial cells were lymphocytes of the suppressor/cytotoxic phenotype (CD 8+), which was also the most prominent cell type in the normal mucosal stroma. Significant differences were observed for the content of CD 8-, OKM 1-, and CD 4-positive cells in the epithelium of normal oral mucosa, SCC, and BCE, and for the amount of CD 1-positive Langerhans cells in the connective tissue of the different groups of tissues. When CD 4/CD 8 ratios were calculated, differences between SCC and BCE became most evident. A CD 4/CD 8 ratio greater 0.5 was seen to be characteristic for BCE. Thus, in contrast to the striking preponderance of suppressor/cytotoxic lymphocytes (CD 8+) in SCC, BCE showed typically almost balanced numbers of suppressor/cytotoxic (CD 8+) and helper/inducer (CD 4+) lymphocytes. This finding further underlines the biological differences recognized between these most common neoplasias of the head and neck.     

Expression of CDIa on monocytes cultured with supernatants from periodontally diseased gingival epithelial cells

OBJECTIVE: Langerhans cells are believed to originate from the monocyte lineage and have been reported to increase in number with plaque accumulation and gingival inflammation. The aim of this study was to investigate the effects of local gingival epithelial factors on the induction of CDla, a Langerhans cell phenotype, on monocyte rich populations.
MATERIALS AND METHODS: Peripheral blood monocyte rich populations from healthy subjects were cultured for 24 h with either healthy gingival or periodontally diseased gingival epithelial supernatants. Additionally, the monocyte rich populations were cultured with cytokines IL-Iα, IL-Iβ, IL-6 and TNF-α which are known to be produced by epithelial cells or co-cultured with autologous epithelial cells. The percent CDIa positive cells was determined using FACS analysis.
RESULTS: Healthy gingival supernatants did not induce CDIa expression in monocyte rich populations, however, a significant increase in per cent CDla⁺ cells for monocyte rich populations cultured with five (P < 0.01) of six periodontal gingival epithelial supernatants was found. IL-lα or TNF-α (10ng/well) resulted in a significant increase in the per cent CDla⁺ cells (P < 0.01). Depletion of CDla⁺ Langerhans cells from healthy gingival epithelium did not enhance induction of CDIa expression in monocyte rich populations. Monocyte rich populations cultured together with non-depleted epithelial cultures resulted in a decreased percent of CDla⁺ cells.
CONCLUSION: These findings indicated that epithelial factor/s associated with periodontally involved epithelia, may be involved in inducing a Langerhans cell phenotype in monocyte rich populations. The data also provide indirect evidence for a role of Langerhans cells in inhibiting induction of CDIa in healthy epithelium.     

New views on the construction of human gingival epithelium

The effects on the basal and spinous layers of human keratinized oral epithelium of 2 glutaraldehyde-based fixatives with buffers hypoosmolar and isoosmolar to blood, respectively, have been investigated. The first-mentioned solution produced an electron microscopic image corresponding to the classical view, that is an epithelium consisting of closely-packed cells having short and stubby membrane projections and separated by an extremely narrow intercellular space. In the Langerhans cells, the specific granules appeared racket-shaped and had a unilaminar limiting membrane with a periodic structure along its internal face. There are strong reasons to believe that these morphological characteristics are swelling artifacts. The last-mentioned fixative produced keratinocytes provided with numerous microvilli and membrane ruffles and disk-shaped Langerhans cell granules surrounded by a trilaminar membrane. Concomitantly, the intercellular space appears very wide and there is evidence for the view that this is a more realistic picture of the in vivo situation and not a gross distortion caused by shrinking during the tissue processing. Broad interstices can explain certain basic events as rapid cell motility within the epithelium and offer efficient pathways for rapid transport of substances.     

Langerhans cells in oral epithelium of chronically inflamed human gingivae

This study describes the histopathological features and the distribution of oral epithelial Langerhans cells in 19 gingival biopsies originating from an adult Tanzanian population characterized by very poor oral hygiene and severe gingival inflammation. Light-microscopically, all biopsies contained often large inflammatory connective tissue infiltrates, 6 of which predominantly contained plasma cells while the rest were dominated by lymphocytes. Seven specimens contained peculiar accumulations of round lymphoid and dendritic cells in the lower cell layers of the oral epithelium. These phenomena have not previously been demonstrated in human gingiva and deserve further attention in studies on the pathogenesis of periodontal diseases. Immuno-histochemical staining with OKT6, OKT4 and OKT8 antibodies showed markedly increased numbers of OKT6-positive cells in 7 specimens and clusters of OKT4- and OKT8-positive cells in the oral epithelium of 4 specimens. High numbers of OKT6-positive cells were not related to the presence of intra-epithelial, non-keratinocyte infiltrates or large connective tissue infiltrates. The variable numbers of oral epithelial Langerhans cells may therefore result from different bacterial antigens elucidating different responses or, alternatively, reflect different responses to similar plaque antigens penetrating the surface of the oral epithelium.     

Extent and diversity of inflammatory cell infiltrates in squamous cell carcinomas and basal cell epitheliomas of the head and neck

Using monoclonal antibodies reactive with Langerhans' cells (LCs), macrophages, and T cell subpopulations, the density and proportions of cells of the immune system of the normal oral mucosa were determined immunohistochemically, and compared with findings in oral squamous cell carcinomas (SCC) and basal cell epitheliomas (BCE). In normal oral epithelia, the dominant cell type was the LC, positive for CD 1, and expressing HLA-DR antigens (DR⁺). Many intraepithelial cells were lymphocytes of the suppressor/cytotoxic phenotype (CD 8⁺), which was also the most prominent cell type in the normal mueosal slroma. Significant differences were observed for the content of CD 8-, OKM 1-, and CD 4-positive cells in the epithelium of normal oral mucosa, SCC, and BCE, and for the amount of CD 1-positive Langerhans cells in the connective tissue of the different groups of tissues. When CD 4/CD 8 ratios were calculated, differences between SCC and BCE became most evident. A CD 4/CD 8 ratio greater 0.5 was seen to be characteristic for BCE. Thus, in contrast to the striking preponderance of suppressor/cytotoxic lymphocytes (CD 8⁺) in SCC, BCE showed typically almost balanced numbers of suppressor/cytotoxic (CD 8⁺) and helper/induced (CD 4⁺) lymphocytes. This finding further underlines the biological differences recognized between these most common neoplasias of the head and neck.