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.     

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.     

Cytolethal distending toxin damages the oral epithelium of gingival explants

The cytolethal distending toxin (Cdt), expressed by the periodontal pathogen Aggregatibacter actinomycetemcomitans, inhibits the proliferation of cultured epithelial cells by arresting the cell cycle. The gingival epithelium is an early line of defense against microbial assault. When damaged, bacteria collectively gain entry into underlying connective tissue where microbial products can affect infiltrating inflammatory cells, leading to the destruction of the attachment apparatus. Histological evaluation of rat and healthy human gingival tissue exposed ex vivo to the Cdt for 36 and 18 hours, respectively, revealed extensive detachment of the keratinized outer layer and distention of spinous and basal cells in the oral epithelium. Treated human tissue also exhibited disruption of rete pegs and dissolution of cell junctions. Cells in the connective tissue appeared unaffected. Primary gingival epithelial cells, but not gingival fibroblasts, isolated from the same healthy human tissue were cell-cycle-arrested when treated with the toxin. These findings provide new evidence that the Cdt severely damages the oral epithelium, ex vivo, by specifically targeting epithelial cells, in situ. The Cdt shows preferential targeting of the epithelium as opposed to connective tissue in animal and human gingival explant models. Abbreviations: cytolethal distending toxin (Cdt), connective tissue (CT), 4',6-diamidino-2-phenylindole (DAPI), human gingival epithelial cells (HGEC), human gingival explants (HGX), human gingival fibroblasts (HGF), junctional epithelium (JE), oral epithelium (OE), rete pegs (RP), sulcular epithelium (SE).     

口腔鳞癌中树突状细胞的免疫组化分析

目的　通过分析口腔鳞癌中浸润性树突状细胞的特征性表型,以探讨其在肿瘤微环境中的功能状态。方法　选择未经任何非手术治疗的初发口腔鳞癌患者标本34例作为实验组,30例口腔正常粘膜组织作为对照组。通过免疫组化技术标记组织中树突状细胞(DC)的CD1a,HLA-DR和CD83抗原,观察两种组织中的DC表达3种抗原的状况,结果进行统计学分析。结果　所有病例均未见明显CD83+DC,但均有CD1a+DC,其在口腔鳞癌组织中的浸润程度低于正常口腔粘膜组织,差异有显著性(P<0·05)。在口腔鳞癌组中有27例癌实质内的DC表达HLA- DR抗原,其HLA-DR的阳性表达率为79·41%。结论　口腔鳞癌组织中的DC,其浸润程度下降并存在功能成熟障碍。     

In situ characterization of mononuclear cells in human chronic marginal periodontitis using monoclonal antibodies

Acetone fixed cryostat sections from 25 patients with adult chronic marginal periodontitis were characterized using an indirect immunofluorescence technique with monoclonal antibodies. The amount of B lymphocytes (Leu-12 positive) varied considerably between the specimens and were usually seen in largest numbers in the most apical parts of the cellular infiltrates beneath the pocket epithelium (PE). Varying amounts of T lymphocytes (OKT 3 positive) were demonstrated in all specimens. The amount of T helper cells (OKT 4a positive) exceeded that of T suppressor/cytotoxic cells (OKT 8 positive) in the cellular infiltrates beneath the PE (OKT 4a/ OKT 8 =1.13). There was a more even distribution of these cell types beneath the oral gingival epithelium (OGE). Langerhans cells were observed within and occasionally subjacent to the OGE. Scattered macrophages (Leu-M3 or OK Ia 1 positive) were observed in the inflammatory cell infiltrates and on the connective tissue papillae beneath the OGE. HLA-DR antigen reacting with OK Ia 1 was present on cells corresponding to OKT 6 positive cells in the OGE and subjacent to the OGE as well as in the inflammatory cell infiltrates beneath the PE and in the perivascular infiltrates. In some specimens HLA-DR antigen was also found to be associated with keratinocytes in the outer parts of the OGE. Occasional NK cells (Leu-7 positive) were localized inside and subjacent to the OGE. There was a considerable variation with respect to the number and distribution of the various mononuclear cells between specimens and from section to section from the same specimen.     

口腔黏膜上皮细胞和成纤维细胞的复合培养

目的 为癌前病变临床病理研究建立一种简便、迅速和有效的口腔黏膜上皮细胞和成纤维细胞复合培养的方法,实现体外模拟组织工程化口腔黏膜的发生发展。方法 用DispaseⅡ分离上皮和皮下组织,用KGM培养口腔黏膜上皮细胞。用细胞培养法和组织块培养法获取验用的口腔黏膜上皮细胞和成纤维细胞,并采用复合培养法对两种细胞进行共同培养。结果 用DispaseⅡ可成分地分离上皮和皮下组织。KGM可明显促进口腔黏膜上皮细胞的分裂繁殖。复合培养的HE染色切片显示薄层的结缔组织之上有方形的基底细胞、颗粒细胞和角化层。结论 KGM可明显促进口腔黏膜上皮细胞的分裂和成熟。采用组织培养法获取原代成纤维细胞是适合口腔黏膜取材等特点的有效方法。气液相培养的口腔黏膜下结缔组织可促进上皮细胞的分层和分化。     

Reduced numbers of Langerhans cells and increased HLA-DR expression in keratinocytes in the oral gingival epithelium of HIV-infected patients with periodontitis

BACKGROUND: HIV-seropositive (HIV+) patients become increasingly susceptible to periodontal diseases as HIV infection proceeds. We have previously shown that HIV+ patients with chronic marginal periodontitis (CMP) have remarkably increased numbers of gingival plasma cells in the connective tissue underlying the oral gingival epithelium, but depressed specific serum IgG levels towards periodontopathogenic bacteria. Langerhans cells (LC) and keratinocytes (KC) are antigen-presenting cells that are important in promoting immune responses. METHOD: In this study we examined, by means of immunofluorescence, the distribution and numbers of LC and activated KC in biopsies taken from inflamed periodontal sites in HIV+ and HIV patients with CMP. RESULTS: In the pocket epithelium in both patient groups, basal layer KC expressed HLA-DR molecules. In the oral gingival epithelium of HIV+ patients, basal layer KC also expressed HLA-DR molecules and numbers of LC were decreased as compared with HIV persons. CONCLUSION: The findings suggest that the oral gingiva in HIV+ patients may be affected by inflammation.     

Macrophages and lymphocyte subpopulations in nifedipine- and cyclosporin A-associated human gingival overgrowth

BACKGROUND: Nifedipine and cyclosporin A (CsA) induce gingival overgrowth. Both drugs have immunomodulating effects. It has been suggested that altered immune response is associated with drug-induced gingival overgrowth. In this study, we evaluated whether there were differences in macrophages and lymphocyte subpopulations in human nifedipine- and CsA-associated gingival overgrowth as compared with those in normal gingiva. METHODS: Biopsy samples of overgrown gingiva were obtained from 9 nifedipine-treated cardiac outpatients, 13 CsA-treated renal transplant recipients including 9 patients who were also receiving nifedipine, and 30 healthy control individuals undergoing dental treatment. Serial 5 microm thick cryostat sections were stained with mAbs for CD20 (B-pan), CD68 (macrophages), CD4 (T-helper/inducer), and CD8 (T-cytotoxic/suppressor) using an avidin-biotin-horseradish peroxidase complex method. Numbers of mAb-labeled and all nucleated cells were determined in 3 areas: the connective tissue beneath the sulcular epithelium, the middle connective tissue, and the connective tissue beneath the oral epithelium. Distributions of each type of cell were expressed as percentages of mAb-labeled cells in relation to total number of nucleated cells in a counting zone. Significances of differences between groups were tested by means of the Kruskal-Wallis test, and between pairs of results by means of the Mann-Whitney U-test. RESULTS: The proportion of CD8-labeled cells was significantly higher in connective tissue beneath the sulcular epithelium in the nifedipine group than in the controls (P = 0.014). In both medicated groups, the proportions of CD68-labeled cells were higher in all counting zones than in the controls, but statistically significantly only in the nifedipine group in the connective tissue beneath the oral epithelium (P = 0.008). No intergroup differences were found with respect to CD4- and CD20-labeled cells. The CD4/CD8 ratio was significantly lower in connective tissue beneath the sulcular epithelium in the nifedipine group than in the controls (P= 0.013). CONCLUSION: The results support the idea that immune response may be altered in drug-induced gingival overgrowth.     

Permeability of rodent junctional epithelium to exogenous protein

It has generally been assumed that oral sulcular epithelium, being nonkeratinized, is a vulnerable region which allows bacterial products to pass from the gingival sulcus to the subjacent connective tissue. It has also been suggested that inducing keratinization of the oral sulcular epithelium might create a better barrier to such material. However, this approach ignores the effect of a permeable junctional epithelium, for if exogenous material penetrates this epithelium then the presence of keratinization may be unimportant. Rats possess an orthokeratinized oral sulcular epithelium and so represent what might be considered as an ideal sulcus lining. The protein tracers horseradish peroxidase or microperoxidase were instilled into the gingival sulcus of anesthetized rats. After 1 h, the animals were killed and fixative applied topically and by perfusion. The mandibles were detached, decalcified in EDTA to permit removal at the gingival attachment and sectioning, reacted with diaminobenzidine and H₂O₂ to visualize the tracer, and prepared for light and electron microscopic examination. Controls consisted of: a) animals that had not been treated with horseradish peroxidase, and b) horseradish peroxidase-treated animals incubated without H₂O₂. Microscopic examination revealed penetration of the tracers through the junctional epithelium and into the underlying connective tissue, but never through the adjacent oral sulcular and gingival epithelium. These results suggest that material placed in the sulcus can enter the connective tissue via the junctional epithelium even when the adjacent oral sulcular epithelium forms a keratinized barrier.     

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.     

Experimental gingivitis in the monkey

Rhesus monkeys receiving an oral hygiene program which included brushing, interdental cleansing and topical applications of chlorhexidine gluconate demonstrated a clinically normal gingiva for periods of up to 3 months. Wide fluctuations within individual dental units were noted with respect to histological sulcus depth, degree of connective tissue infiltration with lymphocytes and plasma cells, and total leukocyte or polymorphonuclear leukocyte (PMN) counts in the Junctional epithelium, even when the plaque and gingivitis index scores were 0. Of 18 clinically normal dental units sampled, only 6 appeared free of connective tissue inflammation. However, more than half of the tissue blocks obtained from dental units with a gingivitis and plaque index of 0 also showed scores of 0 with respect to the connective tissue inflammation (CTI) score and the number of PMNs in the Junctional epithelium. Within three days following discontinuation of oral hygiene procedures, rhesus monkeys developed a clinically noticeable gingivitis which began in the interdental papillae. Increases in CTI scores and number of leukocytes in the Junctional epithelium were evident after 2 days without oral hygiene. These values tended to increase further during the experimental period. A slight, but significant increase in sulcus depth was also noted during this time period. Regardless of the clinical state of the gingiva, a positive correlation was established between CTI scores and the number of PMNs and leukocytes in the Junctional epithelium. In early gingivitis, the plasma cells did not appear to outnumber lymphocytes, as has been reported for chronic gingivitis of longer duration.     

Immunofluorescent localization of soluble dental plaque components in human gingiva affected by periodontitis

In order to determine if soluble plaque antigens are found in the gingiva in the course of periodontal disease, plaque was collected from 16 subjects with periodontitis, an extarct of the plaque was prepared, and antisera to the extarct were induced in rabbits; the sera were conjugated with fluorescein isothiocyanate and used in direct immunofluorescent staining of gingival biopsies from the same subjects. Fluorescent staining suggestive of soluble plaque substances were found in all biopsies, most commonly intracellularly in mononuclear cells of the connective tissue. The most common locations of the cells were subjacent to sulcular epithelium, deeper in the connective tissue in chronic inflammatory infiltrates or near vessels, or close to the oral epithelium. Occasionally a fluorescent cell could be seen within a vessel lumen. These localizations possibly represent engulfed materials within macrophages. Other morphologic appearances of fluorescence were seen that might represent immune complexes. The number of fluorescent sites varied among the subjects and seem to be related to the degree of clinical inflammation. Similar results were found using biopsies from subjects who did not contribute plaque for preparing antisera, indicating that the plaque constituents were not specific to an individual.     

The three-dimensional morphology of the epithelium-connective tissue interface of the gingiva as related to age and sex

abstract – The morphology of the epithelium-connective tissue interface was studied in gingival biopsy specimens of 29 male and 16 female Caucasians. Ten were 7–13 years of age, twenty 20–25, and fifteen 65–77 years of age. Separate three-dimensional wax models (200 ×) were produced of the epithelium and connective tissue of the oral aspect of the free and attached gingiva. The average height, width, and number of connective tissue papillae or ridges and the average width of epithelial ridges were assessed for each specimen. In ten specimens of each age group measurements were made of the contact area between the epithelium and connective tissue. The length of the basement membrane and the volume of the epithelium was assessed within the same area. The essential age change of the epithelium-connective tissue interface is the conversion of the connective tissue ridges to papillae. The contact area between the epithelium and connective tissue increases from the prepuberty to 20–25 years of age. The volume of the epithelium does not change with age. Sex-related variations could not be observed.     

Localization of interleukin-1 (IL-1) mRNA-expressing macrophages in human inflamed gingiva and lL-1 activity in gingival crevicular fluid

The exact cell type and site(s) involved in interleukin-1 (lL-1) production during gingival inflammation was determined by combining immunohistochemistry and in situ hybridization. IL-1 messenger RNA (mRNA)-expressing cells in human inflamed gingiva were identified as macrophages. The rate of IL-α mRNA expression in these macrophages was the same as IL-1 β mRNA expression. The rate of IL-1 mRNA expression was higher in connective tissue furthest from the pocket epithelium, although more macrophages were present at the connective tissue subjacent to the pocket epithelium. The IL-1 activity in gingival crevicular fluid (GCF) obtained from inflamed gingiva was higher than that from healthy gingiva and decreased after periodontal therapy. The IL-1 activity in GCF was almost completely abolished by the addition of anti-IL-1α antibody but not by anti-IL-1 β antibody, indicating that IL-1α is the predominant form in GCF. However, the IL-1 activity in GCF was unrelated to the number of IL-1 mRNA-exprerssing macrophages in the same gingival site where the GCF was obtained at the same time. The results suggest that macrophages in the connective tissue subjacent to the oral epithelium contribute to the production of IL-1 but those in connective tissue subjacent to the pocket epithelium play a different role in the generation of gingival inflammation.     

Distribution of ICAM-1, LFA-3 and HLA-DR in healthy and diseased gingival tissues

Cell adhesion molecules are involved in recognition and effector aspects of the host response, including the control of migration of leukocytes into inflammatory sites. In this study we have demonstrated that the distribution of three cell-surface molecules involved in cell interactions, ICAM-1, LFA-3 and HLA-DR is distinct and different in healthy and diseased gingival tissue. ICAM-1 was consistently expressed by junctional epithelial cells in healthy gingiva and by pocket epithelium in diseased gingiva but was not detectable on the majority of keratinocytes in external gingival epithelium. ICAM-1 was also expressed by endothelial cells of gingival blood vessels and a subset of leukocytes in the infiltrated connective tissue in both healthy and diseased gingiva. HLA-DR and LFA-3 were also expressed by epithelial cells and endothelial cells but in patterns which were distinctly different from ICAM-1.     

In-vitro permeability of porcine oral mucosa after epithelial separation, stripping and hydration

The permeability of porcine skin, gingiva, floor of mouth and buccal mucosa was measured in perfusion chambers using isotopically-labelled water and horseradish peroxidase. Values obtained for the permeability of the epithelium of each of these regions, after separation from the connective tissue with EDTA, did not differ significantly from those obtained for the intact tissue; however, the connective tissue alone had a permeability 2-8 times greater than that of the whole tissue. Stripping the surface layers of the floor of mouth mucosa increased its permeability to that of connective tissue. These results indicate that the functional permeability barrier of the oral mucosa, like that of skin, is located in the epithelium and occupies the superficial layers. After exposure to an aqueous environment for up to 67 h, the permeability of skin and keratinized oral mucosa showed similar but slight increases whereas that of non-keratinized mucosa showed a more rapid rise. These differences may reflect the different composition of the intercellular permeability barrier in keratinized and non-keratinized oral tissues.     

Macrophage subpopulations in gingival overgrowth induced by nifedipine and immunosuppressive medication

BACKGROUND: The immunomodulating effects of both immunosuppressive and nifedipine medication have been associated with drug-induced gingival overgrowth. The aim of the study reported here was to evaluate the presence of macrophage subpopulations in normal human gingiva and in gingival overgrowth induced by nifedipine and immunosuppressive medication. METHODS: Gingival samples were taken from 11 nifedipine-medicated cardiac outpatients (nifedipine group), 11 triple-medicated organ-transplant recipients also taking nifedipine (immunosuppression plus nifedipine group), 12 triple-medicated organ-transplant recipients (immunosuppression group), and 20 generally healthy individuals (control group). Cryostat sections were stained with mAbs for inflammatory 27E10, reparative RM3/1, and resident 25F9 macrophages using an avidin-biotin enzyme complex method. Total numbers of mAb-labeled cells were determined in connective tissue beneath sulcular epithelium, connective tissue beneath oral epithelium, and middle connective tissue. Expression of 27E10 was determined in keratinocytes in the oral epithelium. Statistics analyses were undertaken using the chi-square test, the Mann-Whitney U test, the independent samples t test, analysis of variance, and analysis of covariance. RESULTS: Greater numbers of inflammatory 27E10-positive macrophages were found in all 3 medicated groups and counting zones than in the control group except in connective tissue beneath sulcular epithelium in the immunosuppression group. The incidence of specimens expressing 27E10 antigen throughout the oral epithelium was significantly higher in the immunosuppression group (8 of 12) than in the control group (4 of 20) and the nifedipine group (2 of 11). Numbers of reparative RM3/1-positive macrophages were significantly greater in the immunosuppression group in connective tissue beneath oral epithelium than in the control group. The effect was markedly associated with degree of inflammation. Numbers of resident 25F9-positive macrophages were lower in connective tissue beneath sulcular epithelium in the immunosuppression group, and higher in middle connective tissue in the nifedipine group than in the control group. CONCLUSION: Our results show that the nature of drug-induced gingival overgrowth differs somewhat between immunosuppressive and nifedipine medications.     

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.