T-cell costimulatory capacity of oral and skin epithelial cells in vitro: presence of suppressive activity in supernatants from skin epithelial cell cultures

Oral Langerhans cells (LC) have better T-cell costimulatory capacity than skin LC. In this study factors affecting this capacity have been assessed in a mixed epithelial cell lymphocyte reaction (MELR) assay. Flow cytometry analysis of freshly recovered cells revealed major histocompatibility complex (MHC) class II molecule expression on 7.5% of the oral epithelial cells and 9.7% of the skin epithelial cells. Monoclonal anti class II antibodies significantly reduced the T-cell proliferation in the MELR. Pretreatment of skin epithelial cells with interleukin-1beta, tumour necrosis factor-alpha or interferon (IFN)-gamma did not affect the MELR proliferation, but incubation with IFNgamma significantly suppressed the T-cell response. Transfer of supernatants from cultures of skin epithelial cells and allogeneic T cells to cultures of oral epithelial cells and T cells resulted in a reduced T-cell proliferation while supernatants from oral epithelial cells and T cells did not reduce proliferation. The higher proliferation in cultures of T cells and oral epithelial cells than in cultures containing skin epithelial cells may be due to the presence of a suppressive factor in the skin epithelial cell suspensions.     

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.     

Expression of CD1 and HLA-DR by Langerhans cells (LC) in oral lichenoid drug eruptions (IDE) and idiopathic oral lichen planus (LP)

Numbers of Langerhans ceils (LC) expressing the common thymocyte antigen (T6/CD1) are similar in oral lichen planus (LP) and in normal oral epithelium: however, expression of class II major histocompatibility antigens (HLA-DR/Ia) by Langerhans cells is greater in lichen planus than in normal epithelium, a phenomenon believed to be associated with activation and antigen presentation. This study quantified the numbers of T6+ve and HLA-DR + ve Langerhans cells in oral lichen planus and lichenoid drug eruptions (LDE) to investigate whether differences may reflect differing routes of antigen presentation. Six patients with oral lichenoid drug eruptions and six control idiopathic oral lichen planus patients had lesional biopsies. An immunoperoxidase technique was used to demonstrate binding of T6 and HLA-DR antibodies to identify dendritic intra-epithelia! cells as Langerhans cells and activated Langerhans cells, respectively. In lichenoid drug eruptions, the number of HLA-DR+ve LC was significantly lower than the number of T6+ve LC ( P < 0.05), whereas in idiopathic lichen planus the numbers of T6+ve and HLA-DR+ve LC did not differ significantly ( P = 0.20). The results provide evidence for differences in the routes of antigen presentation in lichenoid drug eruptions and idiopathic lichen planus.     

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.     

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.     

Macrophages, dendritic cells and T lymphocytes in rat buccal mucosa and dental pulp following 5-fluorouracil treatment

Cancer chemotherapeutic drugs may affect immunocompetent cells of oral soft tissues, causing an impaired capacity to induce immune defence reactions. This study was designed to investigate changes in the number of macrophages, dendritic cells and T lymphocytes in the oral mucosa and dental pulp following treatment with the antineoplastic agent 5-fluorouracil (5-FU). Rats were given 5-FU (30 mg/kg or 50 mg/kg) i.v. on days 0, 1, 2, 5, 6 and 7. The number of cells in buccal epithelium and dental pulp expressing ED2, MHC class II, or CD2 molecules was analyzed following immunohistochemical peroxidase staining. Major histocompatibility complex (MHC) class II molecules were analyzed in epithelial sheets and in epithelial cell suspensions by flow cytometry. Increasing concentrations of 5-FU changed the morphology of the epithelial Langerhans cells with a reduced dendritic appearance as the most prominent feature. At 50 mg/kg of 5-FU, the oral epithelium detached from the connective tissue at the basement membrane. MHC class II molecule-expressing cells were reduced in number in the lamina propria of the buccal mucosa and in the dental pulp after both low and high dose of 5-FU, but only after high dose in the epithelium. The number of ED2- and CD2-expressing cells in the dental pulp was only slightly reduced by 5-FU treatment at both low and high dose, while these cells decreased in number in the oral mucosa. The varying sensitivity to 5-FU by macrophages, dendritic cells, and T cells depending on the tissues in which they reside may be due to differences in cell origin or differences in antigenic load.     

Characterization of antigen-presenting cells in human apical periodontitis lesions by flow cytometry and immunocytochemistry

AIM: To analyse phenotypic characteristics of antigen-presenting cells (APC), isolated from human periapical lesions by flow cytometry and immunocytochemistry. METHODOLOGY: Sixteen periapical lesions were digested for 15 min with 0.05% collagenase. Mononuclear cells, separated from other inflammatory cells by density centrifugation, were processed for flow cytometry and/or immunocytochemistry. Single and double immunostainings were performed using monoclonal antibodies specific for human CD45, CD3, CD19, CD14, HLA-DR, CD1a, CD83 and CD123. RESULTS: Antigen-presenting cells (HLA-DR(+) cells) represented 32.9 +/- 17.8% of total mononuclear cells. Amongst them, B cells (HLA-DR(+) CD19(+)) were the predominant APC population, followed by activated macrophages (HLA-DR(+) CD14(+)), dendritic cells (DC) (HLA-DR(+) CD14(-) CD19(-) CD3(-)) and activated T cells (HLA-DR(+) CD3(+)). Based on the predominance of T cells (CD3(+)) or B cells and plasma cells (CD19(+) and CD19(lo), respectively) amongst mononuclear cell infiltrates, lesions were divided into T- and B-types. The percentage of DC in T-type lesions (27.1 +/- 6.8% of total HLA-DR(+) cells) was higher, compared with B-type lesions (10.3 +/- 5.2%) (P < 0.01). Within the DC population, the percentages of CD1a (Langerhans cell type) and CD123 (probably plasmacytoid DC type) did not differ significantly between the groups (P > 0.05). However, the percentage of mature DC (CD83(+)) was significantly higher in T-type periapical lesions (P < 0.05). CONCLUSIONS: Flow cytometry and immunocytochemistry are suitable methods for phenotypic analysis of APC after their isolation from human periapical lesions. APC, that were phenotypically heterogeneous, constituted a significant component of infiltrating cells. Lesions with the predominance of T cells were characterized by a higher proportion of mature DC (HLA-DR(+)CD83(+) cells) than lesions with predominance of B cells/plasma cells.     

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.     

高迁移率族蛋白N2抑制口腔鳞状细胞癌增殖作用的体外研究

目的 以人口腔鳞状细胞癌细胞株Tca8113为实验模型,初步探讨高迁移率族蛋白N2（HMGN2）抗口腔鳞状细胞癌的作用。方法 大量培养重组人HMGN2表达载体大肠杆菌BL21,用异丙基-1-硫代-β-呋喃半乳糖苷（IPTG） 诱导HMGN2的表达,产物用B-PER GST Fusion Protein Purification Kit进行纯化。在细胞培养基中加入不同质量浓度的HMGN2,通过MTT、Hoechst 33342荧光染色、流式细胞术和Western-blot法检测其凋亡效果及抗凋亡的分子机制。结果 经MTT检测证明HMGN2能显著抑制Tca8113细胞的生长,通过Hoechst 33342荧光染色、流式细胞术和 Western-blot检测证明HMGN2能使Tca8113的细胞形态发生改变,并且能使Tca8113细胞阻滞于细胞周期的S期,促进Tca8113细胞凋亡。结论 HMGN2可以促进人口腔鳞状细胞癌细胞的凋亡。     

Langerhans cells from oral epithelium are more effective in stimulating allogeneic t-cells in vitro than Langerhans cells from skin epithelium

Dendritic cells, such as Langerhans cells (LC), in different ectodermal compartments may have different functional capabilities. The present study was undertaken to compare oral Langerhans cells (LC) with those of the epidermis in terms of their ability to co-stimulate T-cells in vitro. A Mixed Epithelial Cell Lymphocyte Reaction (MELR) and a mitogen-driven (concanavalin A) T-cell proliferation assay were used. In both assays, LC in a crude cell suspension of freshly isolated oral epithelial cells were found to be five times more effective in mediating T-cell proliferation than freshly isolated epidermal LC. Twenty-four-hour cell culture at 37 degrees C enhanced the T-cell response in the MELR compared with cells cultured at 4 degrees C. This applied to both skin and oral epithelial cells. Oral and skin epithelial cell suspensions depleted of LC lost the capacity to stimulate allogeneic T-cells. Incubation of the epithelial cell suspensions with recombinant Granulocyte/Macrophage-Colony Stimulating Factor (rGM-CSF) did not enhance the co-stimulating capacity of the LC. Titration of different numbers of oral and skin LC to T-cells showed that skin LC were never able to reach more than 44% of the maximal stimulatory capacity of oral LC. Data show that oral LC are more efficient than skin LC in providing co-stimulatory signals to T-cells, suggesting a difference in functional capacity between the two cell populations.     

Gingival keratinocytes express HLA-DR antigens in chronic gingivitis

The expression of the histocompatibility antigens HLA-DR and HLA-A, B, C within periodontally diseased tissue was investigated using immunohistological and histochemical techniques. Tissue was obtained from 18 patients with periodontal disease and from 2 healthy volunteers. HLA-DR antigen was expressed by the keratinocytes of the oral epithelium in all inflamed samples but was not a feature of normal tissue where HLA-DR reactivity was confined to Langerhans cells. These results are consistent with an underlying cellular immune process. Using a variety of phenotypic markers it was possible to characterize the macrophage population within the connective tissue into 2 distinct types: an antigen-presenting cell type located subjacent to the oral epithelium and a phagocytic cell type situated deep within the connective tissue.     

Gingival keratinocytes express HLA-DR antigens in chronic gingivitis

The expression of the histocompatibility antigens HLA-DR and HLA-A, B, C within periodontally diseased tissue was investigated using immunohistological and histochemical techniques. Tissue was obtained from 18 patients with periodontal disease and from 2 healthy volunteers. HLA-DR antigen was expressed by the keratinocytes of the oral epithelium in all inflamed samples but was not a feature of normal tissue where HLA-DR reactivity was confined to Langerhans cells. These results are consistent with an underlying cellular immune process. Using a variety of phenotypic markers it was possible to characterize the macrophage population within the connective tissue into 2 distinct types: an antigen-presenting cell type located subjacent to the oral epithelium and a phagocytic cell type situated deep within the connective tissue.     

Langerhans cell distribution and keratinocyte expression of HLADR in oral lichen planus

Keratinocyte expression of the Class II major histocompatibility complex antigen HLADR, is seen in several inflammatory disorders of skin and mucosa, including lichen planus. The purpose of this study is to determine whether the distribution of Langerhans cells and their expression of CD4 in oral lichen planus is related to keratinocyte HLADR. The numbers of CD1- and CD4-positive Langerhans cells were compared in areas of keratinocyte HLADR and areas showing no expression in oral lichen planus and with normal oral mucosa. Cells were identified using an immunoalkaline phosphatase technique and numbers were expressed per mm epithelial surface length. In lichen planus, an increase both in the number of Langerhans cells and the numbers expressing CD4 were found in areas of keratinocyte HLADR expression compared with HLADR negative areas and with normal oral mucosa. There was no difference in the numbers of Langerhans cells or their expression of CD4 between HLADR-negative areas in LP and normal oral mucosa. These results show that the distribution of Langerhans cells is related to keratinocyte expression of HLADR and suggest that Langerhans cell entry may be enhanced in these areas. Whilst it is possible this enhancement is mediated by CD4/HLADR interaction, other molecules are also likely to be important in controlling Langerhans cell entry into oral mucosa.     

口腔鳞癌中趋化因子受体CXCR4的临床病理及细胞学研究

目的检测CXCR4蛋白在口腔鳞状细胞癌（OSCC）组织和OSCC细胞系中的表达,探讨CXCR4蛋白的表达与OSCC临床病理及SDF-1/CXCR4轴与OSCC细胞增殖的关系.方法用免疫组织化学SP法检测91例OSCC中CXCR4的表达,用细胞培养、免疫组化法检测CXCR4蛋白的表达模式,流式细胞仪直接免疫荧光法测定CXCR4蛋白的表达量,MTT法检测细胞的增殖能力.结果CXCR4在OSCC组织中总阳性表达率为62.6%,与肿瘤的大小、病理分级、淋巴结转移、肿瘤增殖密切相关（P＜0.05）.CXCR4蛋白在OSCC细胞系中亦呈强阳性表达,OSCC细胞在SDF-l作用下增殖反应明显增强,CXCR4抗体可明显抑制肿瘤细胞的增殖.结论CXCR4与肿瘤的大小、病理分级、淋巴结转移及肿瘤增殖密切相关,对评估OSCC的生物学行为有意义.     

三氧化二砷诱导口腔鳞癌细胞凋亡的机制探讨

目的：探讨三氧化二砷诱导口腔鳞癌细胞凋亡的机制。方法：通过对两种舌鳞癌细胞系OSC－19，Tca8113经三氧化二砷作用后电镜下形态学改变、流式细胞仪测得的细胞周期变化、线粒体跨膜电位改变、凋亡相关蛋白BCL－2、p16、p53、Caspase-3及PARP变化；初步探讨三氧化二砷诱导舌鳞癌细胞系凋亡的机制。结果：（1）三氧化二砷抑制口腔鳞癌细胞生长通过毒性损伤和诱导凋亡双重途径来实现；（2）经三氧化二砷作用后，western blot检测bcl-2、p53、p16基因表达蛋白无变化，而caspase-3,PARP发生改变；（3）经三氧化二砷作用后，两类舌鳞癌细胞系线粒体跨膜电位明显下降，细胞周期G2－M期阻滞，且与凋亡产生同步。结论：（1）线粒体和微管可能是三氧化二砷的主要作用位点，为三氧化二砷发挥凋亡诱导效应的启动因素；（2）Caspase-3途径的激活可能为三氧化二砷诱导口腔鳞癌细胞凋亡的关键途径之一。     

Culturing primary human gingival epithelial cells: comparison of two isolation techniques.

BACKGROUND: Cultured epithelial cells offer many potential clinical applications. There have generally been two techniques that have been used to cultivate oral keratinocytes, which include the direct explant technique and the enzymatic method. Little work has been done comparing these two techniques and their capacity to isolate and cultivate oral keratinocytes. Objectives: The objectives of this study were to (1) investigate the difference in the percentage of keratinocyte isolation between the direct explant technique and the enzymatic method of human gingival epithelial cell culture and (2) to examine the effect of age and sex of the subjects providing the tissue samples on (a) the success in cultivation and (b) the growth patterns of gingival keratinocytes. MATERIAL AND METHODS: Gingival tissue was obtained from healthy human subjects and was used for keratinocyte isolation using the direct explant technique or the enzymatic method. Epithelial cell cultures from each of the two culture techniques were selected randomly for flow cytometry analysis for cell expression of vimentin and cytokeratin. Growth rate assays were also conducted. RESULTS: The success rate for cultivation from the direct explant technique was higher (82%) than in the enzymatic method (57.9%). The success rate of both methods was not significantly associated with either age or sex of the subjects providing the tissue. From flow cytometry, the average percentage of cells that was positive to anti-pan cytokeratin was nearly the same for both methods at about 97%. It was noted that the cells from the enzymatic method gave significantly higher percentages of cells that were positive to anti-pan cytokeratin only. CONCLUSION: Both the direct explant technique and the enzymatic method can be used for isolating and culturing human oral keratinocytes. The direct explant technique appeared to be more successful in culturing human oral keratinocytes than the enzymatic method, although there were limitations found with both methods. The age and sex of the subjects providing the gingival samples did not appear to be a factor influencing the success rate in culturing the keratinocytes. However, contamination by oral microbiological flora from the gingival tissue samples remained an ever present problem. Further studies are needed in the investigation of clinical applications of these two epithelial cell isolation methods.     

Antigen presenting capacity of Langerhans cells from rat oral epithelium

The ability of Langerhans cells (LC) from rat oral mucosa to internalize and process antigens and to participate in the induction of T cell mitogenesis was examined. To purify LC from epithelial cells, monoclonal anti-class II antibodies and immunomagnetic beads were employed. Suspensions of epithelial cells, containing LC, were found to be effective in mediating a Con A-induced T cell proliferation. Depletion of class II molecule-expressing LC reduced the proliferation of T cells by 80%. Presentation of ovalbumin (OA) to primed T cells was found to be dependent on the concentration of OA and the number of LC. Partially purified LC were five times as effective in inducing proliferation of primed T cells as the untreated suspension of epithelial cells. The data suggest that LC obtained from rat oral mucosa can generate accessory signals, process antigens and serve as antigen-presenting cells.     

Expression of CDw29 and CD45R antigens on epithelial cells in oral lichen planus

The CD45R and CDw29 antigens are expressed on naive and primed helper T cell populations which serve suppressor-inducer or helper-inducer functions, respectively. These antigens may also be expressed on epithelial cell subpopulations. In the present study, monoclonal antibodies reacting with T lymphocytes and Langerhans cells (LC) were used to characterize the expression of CD45R and CDw29 antigens in oral lichen planus. CDw29 was expressed by LC and lymphocytic cells whereas keratinocyte reactivity varied from negative through to full thickness staining. Expression of CD45R was confined to intraepithelial cells with either lymphocytic or dendritic morphology. A relatively constant ratio of CD1a + LC to CD45R + cells (2:1) was seen. These results demonstrate the existence of intraepithelial cells expressing antigens which are functionally important in T cell responses and which may provide local immunoregulatory influences.     

Invasive front in oral squamous cell carcinoma: image and flow cytometric analysis with clinicopathologic correlation

OBJECTIVE: Pathologists have drawn attention to the invasive tumor front (ITF) in the determination of the biologic aggressiveness of oral cancer. We have attempted to discover the prognostic significance of cancer cells with abnormal DNA content at the ITF of oral squamous cell carcinoma. STUDY DESIGN: A comparative DNA analysis by means of image cytometry and flow cytometry was conducted to confirm the usefulness of image cytometry in detecting cancer cells having abnormal DNA content at the ITF. The prognostic value of cancer cells with abnormal DNA content ws examined by a multivariate analysis for 195 patients with oral squamous cell carcinoma. RESULTS: In the comparative DNA analysis, it was suggested that image cytometry is useful for detecting cancer cells with abnormal DNA content (4c exceeding rate [4cER]), which is associated with poor prognosis of patients with oral squamous cell carcinoma. In the multivariate analysis, 3 independent factors were found to significantly influence cause-specific survival. These are, in decreasing order of influence, (1) abnormal DNA content (4cER), (2) clinical stage, and (3) growth type. CONCLUSION: The presence of cancer cells with abnormal DNA content of the ITF in conjunction with clinical findings (clinical stage and growth type) can give additional useful information when selecting treatment strategies for oral cancer patients.