Characterization of T lymphocyte clones derived from Porphyromonas gingivalis infected subjects

Porphyromonas gingivalis plays a major role in the pathogenesis of periodontal disease, however some individuals with P. gingivalis infection do not experience periodontal breakdown. The aim of this study was to investigate the proliferative responses of two highly defined groups of subjects and to establish and characterize peripheral blood and gingival cell T cell lines and clones from subjects from these groups, The two groups were selected on the basis of P. gingivalis in their plaque and the presence of serum anti-P. gingiralis antibodies. Both groups therefore were seen to have P. gingivalis and to have responded to it. They however differed only in their clinical susceptibility (adult periodontitis) or resistance (gingivitis) to periodontal breakdown. Dose responses of peripheral blood mononuclear cells extracted from the subjects showed a trend towards a lower response by the adult periodontitis group to P. gingivalis outer membrane (OM) antigens. Peripheral blood T cell lines and clones responsive to P. gingivalis OM were established from a high responding gingivitis subject and a low responding adult periodontitis subject. Gingival T cell lines and clones were also derived from cells extracted from the periodontal tissues of the same periodontitis subject. The majority of T cells in the peripheral blood T cell line from the gingivitis subject were CD4 while those from the adult periodontitis subject were CD8. The gingival T cell line was CD3+ve CD4-ve and CD8-ve. All lines and clones proliferated slowly to P. gingivalis OM but phytohaemagglutinin (PHA) induced an increase in DNA synthesis in those derived from the gingivitis subject with little to no effect on those established from the adult periodontitis subject. Furthermore. PHA inhibited the proliferative response of the CD8 clone derived from the adult periodontitis subject. Phenotypic analysis demonstrated that all the peripheral blood clones expressed the αβ TCR while the gingival T cell clones expressed the γδ TCR. All clones had the memory/primed CD45RO+ve phenotype and at least 80% of cells in each clone were HLA-DR + ve. A lower percent of gingival cells expressed CD45RA than the CD4 peripheral blood clones and the two CD8 clones also had a decreased CD45RA expression. The gingival T cell clones also expressed a low percent CD25 as did the CD8 clone derived from the adult periodontitis subject. The results suggest that clones derived from the gingivitis and adult periodontitis subject may be functionally different. The presence of γδ T cells in adult periodontitis remains to be confirmed and their function determined.     

The nature of the inflammatory infiltrates in childhood gingivitis, juvenile periodontitis and adult periodontitis: immunocytochemical studies using a monoclonal antibody to HLA Dr

Routinely fixed and processed gingival biopsies from childhood gingivitis, juvenile periodontitis and chronic adult periodontitis patients have been stained immunocytochemically with a monoclonal anti HLADr antibody to aid in the identification and quantification of cell types in the inflammatory infiltrates. Using immunoperoxidase staining and morphological criteria, 9 cell types were quantified in 30 patients. Lesions in the 3 groups were found to differ widely both in size and composition. In the small childhood gingivitis lesions, most cells were small lymphocytes, over half of which were HLADr positive, whereas in juvenile periodontitis biopsies, well over half the infiltrate was plasma cells. The chronic adult periodontitis samples showed greater variability in composition between these 2 extremes, perhaps reflecting differences in disease activity. These results suggest that, when disease is quiescent, the volume of inflamed gingival connective tissue is small and is dominated by B-small lymphocytes, whilst on activation, the lesion increases in size and much of the B-lymphocyte population is transformed to plasma cells. This view corroborates the results of other workers with regard to juvenile periodontitis, but suggests a different interpretation of the quiescent lesion of childhood gingivitis from that current in the literature.     

Langerhans cells in the gingival epithelium

Langerhans cells (LCs) were specifically demonstrated by monoclonal antibody OKT 6. In healthy gingival tissue LCs were mainly seen in stratum spinosum of the surface epithelium. They had small nuclei and with long cell processes. The LCs of oral epithelium in marginal gingivitis and adult periodontitis tissue were more in numbers than in healthy tissue. In juvenile periodontitis tissue LCs numbers seemed to be increased obviously in comparison to the healthy tissue. The LCs were round and often located in both deep spinous and basal layers. These results demonstrate that LCs play an important role in the local immune response of the periodontal tissues.     

Tissue localization of Actinobacillus actinomycetemcomitans in human periodontitis. I. Light, immunofluorescence and electron microscopic studies

Invasion of periodontal tissues by different bacterial morphotypes has been reported in human periodontitis; however, limited information is available as to prevalence, localization and the bacterial species involved. The present study determined prevalence and gingival localization of Actinobacillus (Haemophilus) actinomycetemcomitans in periodontal lesions of juvenile periodontitis patients. Thirty-five gingival biopsies were obtained from 12 juvenile periodontitis patients at the time of periodontal therapy. One additional control biopsy was obtained from each of two adult periodontally healthy subjects, one adult periodontitis patient and one periodontally healthy monkey (Macaca fosibolius). The biopsies were carefully processed to avoid mechanical introduction of bacteria into the tissues and were examined using light and electron microscopy. Rabbit antisera specific for the three A. actinomycetemcomitans serotypes were used for immunofluorescence microscopic localization of A. actinomycetemcomitans antigens in the gingival sections. Immunofluorescence microscopy showed A. actinomycetemcomitans specific antigens in the gingival tissues of 11 of the 12 juvenile patients examined. None of the control specimens showed evidence of A. actinomycetemcomitans antigens in the gingival connective tissue. One specimen from a periodontally healthy subject and the monkey biopsy, however, showed A. actinomycetemcomitans antigens in bacterial plaque on the surface of the crevicular epithelium. Transmission electron microscopic examination showed microcolonies of small gram-negative rods in the connective tissue, as well as single bacterial cells between collagen fibers and in areas of cell debris. In addition to these extracellular bacterial cells, evidence of bacterial cells was also found within gingival connective tissue phagocytic cells. The data from the present study suggest that the gingival tissue in juvenile periodontitis lesions harbors A. actinomycetemcomitans.     

Vascular expansion in chronic periodontitis

Animal studies have demonstrated expansion and remodelling of gingival blood vessels in inflamed gingival tissues. However, there is a paucity of information, regarding the vascular response in human gingivitis and periodontitis. In this study, gingival biopsies were obtained from 51 separate patients. Fifteen minimally inflamed, 16 gingivitis and 20 periodontitis specimens were studied. Gingival biopsies were divided into five fields, and a quantitative survey of vascular changes was performed. In fields adjacent to the bacterial plaque irritant, vessel profiles were increased in number with the development of the advanced periodontal lesion. The diameter of blood vessels throughout the entire thickness of gingival biopsies was found to increase with advancing periodontal disease. It is concluded that considerable remodelling of the gingival vasculature occurs in chronic periodontitis, and that this may contribute to the tissue destruction seen in this disease.     

Gingival crevice neutrophil function in periodontal lesions

Polymorphonuclear neutrophils (PMN) may play an important role in protection of the host from pathogenic microorganisms associated with periodontal tissue destruction. The purpose of the present study was to test the hypothesis that unusually severe periodontitis may be associated with defective PMN function at the local disease site. The patients studied included young patients with rapidly progressive periodontitis (including juvenile periodontitis), age-matched patients with gingivitis, and older patients with chronic periodontitis. Gingival crevice (GC) PMN were collected from 10 lesions of each patient by a crevicular washing technique. The number and viability of GC PMN recovered were determined. Their phagocytic capacity was assayed in vitro as a percentage of cells capable of engulfing latex particles. No differences were observed between the periodontitis groups in the number or viability of GC PMN recovered. A statistically significant reduction in mean phagocytic capacity was observed in PMN recovered from lesions of rapidly progressive periodontitis when compared with lesions of chronic periodontitis (12.9 ± 2.1 % vs. 83.7 ± 4.8 %). GC PMNs recovered from non-diseased sites of patients with localized juvenile periodontitis did not show this decreased function. These results suggest that locally diminished PMN function in rapidly progressive and juvenile periodontitis is associated with the environment of these lesions.     

Cell-surface proteoglycan expression by lymphocytes from peripheral blood and gingiva in health and periodontal disease.

Cell-surface proteoglycans are involved in lymphocyte migration and activation. This study investigated the expression of syndecan-1, syndecan-4, and glypican in peripheral blood lymphocytes and by lymphocytes in variously inflamed periodontal tissues. Gingival specimens from healthy, gingivitis, or chronic periodontitis sites were stained by means of antibodies against B- and T-lymphocytes and also syndecan-1, syndecan-4, and glypican. Syndecan-1 expression by peripheral blood mononuclear cells (PBMC) from healthy, gingivitis, and chronic periodontitis subjects was assessed by flow cytometry. Syndecan-1 was expressed by B-cells/plasma cells but not T-cells in both gingivitis and chronic periodontitis lesions. Both B-cells/plasma cells and T-cells in gingivitis and chronic periodontitis expressed syndecan-4. Glypican was expressed only by macrophages. Stimulation of PBMC with mitogens and growth factors modulated syndecan-1 expression in both the T- and B-cells. Thus, cell-surface proteoglycan expression by lymphocytes in periodontal inflammation is cell-type-specific and may be modulated by inflammation.     

Assessment of complement cleavage in gingival fluid in humans with and without periodontal disease

The activation of the complement system may be an important immunopathologic mechanism in the initiation and progression of periodontal disease. The purpose of this study was to assess cleavage of complement components C3 (terminal pathway), C4 (classical pathway) and B (alternative pathway) in gingival fluid obtained from patients with varying types and severities of periodontal disease. Gingival fluid samples were obtained on filter paper strips from 18 healthy sites, 16 gingivitis, 59 chronic adult periodontitis, 45 rapidly progressive periodontitis, and 11 juvenile periodontitis lesions. Each patient was categorized on the basis of age and clinical indices, including Gingival Index, Plaque Index, measurement of pocket depth and loss of periodontal attachment in millimeters, presence of suppuration and bleeding on probing. Cleavage of C3, C4, and B from each site was assessed simultaneously by multilayer crossedimmunoelectrophoresis using solid phase absorbed specific antisera. The mean percentage C3 conversion ranged from a low of 12.6% in the healthy to 90.2% in the juvenile periodontitis group. Statistically significant differences, as determined by the Mann-Whitney U-Test, were observed between healthy sites and all other groups, gingivitis and all periodontitis groups, and juvenile vs. chronic periodontitis. C4 was present in all sites examined, but its cleavage product C4c was only observed in sites with juvenile periodontitis. B and its cleavage product Bb were consistently present in gingival fluid from inflamed lesions. The percentage of C3 cleaved to C3c correlated significantly (p < 0.001) with pocket depth (rho=0.58), gingivitis (rho=0.68) and bleeding on probing (rho=0.63). These results suggest that 1) increased complement cleavage is associated with increased severity of inflammation and periodontal destruction, and 2) classical pathway activation does not appear to occur in gingivitis and adult periodontitis, but may occur in juvenile periodontitis.     

Tumor necrosis factor-alpha expression and detection of apoptosis at the site of chronic periodontitis in AIDS patients

BACKGROUND: Apoptosis, or programmed cell death, is associated with the regulation of the life cell cycle of leukocytes in healthy and diseased states. OBJECTIVES: In the present study, we investigated the presence of apoptosis of mononuclear inflammatory cells in the periodontal lesion from adult periodontitis in healthy control patients and AIDS patients. MATERIALS AND METHODS: Tissue samples adjacent to a 5-6 mm gingival sulcus, measured with a periodontal probe, were obtained during routine periodontal surgical procedures. The direct immuno-peroxidase of digoxigenin-labeled genomic DNA method was used for in situ detection of apoptosis in gingival tissues. RESULTS: Many tumor necrosis factor-alpha (TNFalpha)-positive cells, detected by immunohistochemistry method, were observed in gingival samples of both groups of patients. In addition, a significant lower number ( p < 0.05) of mononuclear apoptotic cells were observed in AIDS patients when compared with healthy control patients. CONCLUSION: These data suggested an important role of the apoptosis of mononuclear cells in the pathogenesis of chronic adult periodontitis in AIDS patients.     

In situ hybridization study on tissue inhibitors of metalloproteinases (TIMPs) mRNA-expressing cells in human inflamed gingival tissue

This study presents the exact cell types and localization of tissue inhibitors of metalloproteinases (TIMPs) production sites in periodontal diseased gingiva by means of in situ hybridization. Gingival tissue specimens were fixed, embedded and hybridized in situ with specific digoxigenin-labeled cRNA probes (386 and 496 bp). TIMP-1 and -2 mRNAs were expressed on macrophages, mononuclear cells, capillary endothelial cells and some fibroblasts throughout the gingival tissue. In periodontitis, TIMP-1 and -2 mRNA-expressing cells showed significantly different localization. TIMP-1 mRNA was broadly observed in the gingival connective tissue while TIMP-2 mRNA was predominantly expressed in the connective tissue adjacent to the pocket epithelium (p < 0.01). Fewer TIMPs mRNA were observed in minimal gingivitis than in periodontitis, especially in the middle zone of gingival tissue. Thus, TIMP-1 and TIMP-2 mRNA was detected differentially and site-specifically in periodontal diseased gingival tissue.     

CD29 expression on CD4+ gingival lymphocytes supports migration of activated memory T lymphocytes to diseased periodontal tissue

The cell surface phenotypes of CD4⁺ cells extracted from inflammatory periodontal disease tissues were analyzed using two- and three-color immunofluorescence and flow cytometry. Cells extracted from both adult periodontal and localized juvenile periodontitis lesions showed a depressed CD4/CD8 ratio (1.0±0.1 adult periodontitis and 1.1 ±0.1 localized juvenile periodontitis) compared with cells recovered from normal/marginal gingivitis tissue (1.8 ±0.2) or with normal peripheral blood cells (2.1 ±0.1) or periodontal disease blood cells (2.1±0.1 and 1.7±0.1 for adult periodontitis and juvenile periodontitis, respectively). The monoclonal antibodies anti-2H4 and anti-4B4 were used to identify the CD45RA and CD29 antigens respectively on CD4⁺ T cells from the periodontal disease lesions. In peripheral blood, CD29⁺ cells accounted for 66–77% of the CD4⁺ population, and CD45RA⁺ cells accounted for 22–27% of the CD4⁺ subset. No differences in expression were found between peripheral blood lymphocytes from normal subjects and from periodontal disease patients. Two-color analyses of lymphocytes from periodontal diseased tissues showed that 87–89% of the CD4⁺ population were CD29⁺ and that 70–79% of the CD4⁺ cells were CD45RA⁺. Normal tissues contained significantly fewer CD4⁺CD29⁺ cells (56±4%) and CD4⁺CD45RA⁺ cells (40±4%) on average, and few, if any double-labelled cells could be accounted for. These data implied that a significant percentage of the CD4⁺ cells from the diseased tissues were both CD29⁺ and CD45RA⁺ and that these populations are found in quite different proportions in diseased periodontal tissue than in peripheral blood or nondiseased tissue. In further analyses using three-color cytometry the mean percentage of CD4⁺CD29⁺CD45RA⁺ lymphocytes extracted from periodontal disease lesions was 43±9% of the CD4⁺ population. These results suggest that CD4⁺ T lymphocytes in periodontal disease not only demonstrate varying levels of maturity but also that the accumulation of CD4⁺ T cells within the periodontal tissues maybe a result of increased adhesion and transendothelial migration.     

Isolation of alkaline phosphatase-positive gingival fibroblasts from patients with chronic inflammatory periodontal disease

We have reported recently that increased expression of membrane alkaline phosphatase (ALP) activity is a phenotypical characteristic of gingival fibroblasts located in chronic inflammatory periodontal lesions. To understand the cellular properties of these cells, we isolated ALP-positive gingival fibroblasts from patients with adult periodontitis and evaluated their proliferative potential. Using an enzymatic digestion procedure, we prepared gingival cell suspensions containing ALP-positive fibroblasts without affecting their ALP activities. These cell suspensions were then subjected to 1 g sedimentation, followed by allowing cells to adhere to substrata. Using this procedure, 71.9% of isolated cells were ALP-positive. Dissociation of ALP-positive fibroblasts and contamination by non-fibroblastic cells were examined by cytochemical and immunocytochemical analyses. The proliferative capacity of ALP-positive fibroblasts in culture was assessed by monitoring the proportion of ALP-positive cells after repeated subculture passages and by labelling DNA-synthesizing cells with bromodeoxyuridine (BrdU). The proportion of ALP-positive fibroblasts decreased during cell culture passages without an apparent change in the ALP-positive phenotype. The percentage of BrdU-positive cells was significantly lower among ALP-positive than among ALP-negative fibroblasts. These results indicate that ALP-positive fibroblasts in chronic inflammatory periodontal lesions have low growth potential. We suggest that their reduced capacity to grow in vitro reflects a more differentiated state induced under inflammatory conditions in vivo .     

Lymphokine production by human gingival lymphocytes

A variety of immunological mechanisms of the host have been implicated as contributing factors in the pathogenesis of human periodontal diseases. Studies using peripheral blood mononuclear cells have been equivocal; however, development of a technique for isolating lymphoid cells from human gingival tissues has allowed the investigation of local immune activities. This study was done to determine whether gingival lymphoid cells from subjects with no disease, gingivitis or periodontitis could produce lymphokines directly or in response to stimulation with plaque, Actinomyces viscosus or Bacteroides gingivalis. Results show that cells from non-diseased gingiva produce very low levels of chemotactic factor (CF), leukocyte migration inhibition factor (LIF) or mitogenic factor (MF) directly or in response to the plaque or bacteria. Gingival cells from gingivitis tissues show low levels of lymphokine production when unstimulated, but this increases in response to plaque and A. viscosus. Cells from periodontitis tissues similarly show low to moderate lymphokine levels in unstimulated cultures but an increased production in response to plaque and B. gingivalis. These data suggest that gingival lymphoid cells may be sensitized, and become reactive in vivo in disease states, and that various periodontal disease states may be related to specific oral bacteria.     

Periodontal diseases in the child and adolescent

BACKGROUND: Periodontal diseases are among the most frequent diseases affecting children and adolescents. These include gingivitis, localized or generalized aggressive periodontitis (a.k.a., early onset periodontitis which includes generalized or localized prepubertal periodontitis and juvenile periodontitis) and periodontal diseases associated with systemic disorders. The best approach to managing periodontal diseases is prevention, followed by early detection and treatment. METHODS: This paper reviews the current literature concerning the most common periodontal diseases affecting children: chronic gingivitis (or dental plaque-induced gingival diseases) and early onset periodontitis (or aggressive periodontitis), including prepubertal and juvenile periodontitis. In addition, systemic diseases that affect the periodontium and oral lesions commonly found in young children are addressed. The prevalence, diagnostic characteristics, microbiology, host-related factors, and therapeutic management of each of these disease entities are thoroughly discussed.     

Porphyromonas gingivalis antigen preferentially stimulates T cells to express IL-17 but not receptor activator of NF-kappaB ligand in vitro

Although T cells have been implicated in the pathogenesis and are considered to be central to both their progression and control of chronic inflammatory periodontal diseases, the precise contribution of T cells to tissue destruction has not been fully clarified. Recently, interleukin (IL)-17 and receptor activator of Nuclear factor kappaB NF-kappaB ligand (RANKL) have received much attention as a result of their proinflammatory and bone metabolic roles, respectively. We therefore investigated the effect of outer membrane protein (OMP) from Porphyromonas gingivalis (P. gingivalis) on the expression of IL-17 and RANKL in peripheral blood mononuclear cells (PBMCs) and compared these between gingivitis and periodontitis, which are representative of stable and progressive lesions, respectively. The in situ expression of these molecules was also examined. P. gingivalis OMP stimulated PBMCs to express IL-17 at both the mRNA and protein level. Although the mean expression of mRNA was not different between the two groups, the mean level of IL-17 in the culture supernatants was higher in gingivitis patients than in periodontitis patients. However, the frequency of IL-17-positive samples was higher in the periodontitis patients. This stimulatory effect was not evident for RANKL expression in either periodontitis or gingivitis patients. In gingival tissue samples, IL-17 mRNA was detected in gingivitis more frequently than in periodontitis. The expression of RANKL mRNA was much lower than that of IL-17 in terms of both level and frequency. These results suggest that IL-17 but not RANKL may be involved in the pathogenesis of periodontal diseases. However, there may be negative regulatory mechanisms for IL-17 in gingivitis.     

Toll like receptor 4 and membrane-bound CD14 expressions in gingivitis, periodontitis and CsA-induced gingival overgrowth

Objective

Immune cell recognition of lipopolysaccharides via CD14 and Toll-like receptor 4 (TLR4) complexes plays a crucial role in linking innate and adaptive immune responses. This study was aimed to investigate the expression of TLR4 and membrane-bound CD14 (mCD14) in the gingival tissues of patients with gingivitis, periodontitis and CsA-induced gingival overgrowth.

Design

Gingival tissues were obtained from 10 renal transplant patients receiving cyclosporine-A (CsA) and having gingival overgrowth (GO), 10 patients with chronic periodontitis, 10 generalized aggressive periodontitis, 10 gingivitis and 10 healthy subjects. Immunohistochemistry was performed in order to determine the localization of TLR4 and mCD14 in tissue specimens.

Results

TLR4 and mCD14 expressions were detected in all tissues including healthy gingival biopsies. TLR4 and mCD14 positive cells were predominantly confined to the epithelium–connective tissue interface area, and were highly expressed in the basal cell layer of patients with CsA GO and chronic periodontitis, compared to healthy group (P < 0.05).

Conclusion

The present study suggests that TLR4 and mCD14 protein expressions may be interrelated and appear to be associated with periodontal disease. CsA usage seemed not to affect TLR4 and mCD14 expressions in CsA induced GO tissues.     

Microbiology and management of periodontal infections

The term periodontal disease refers to all diseases that involve the supportive structures of the periodontium. Peridontal diseases commonly begin as a gingivitis and progress to periodontitis. Necrotizing ulcerative gingivitis (NUG) is the most fulminate form of gingivitis. The two main forms of periodontitis are chronic periodontitis (also known as adult periodontitis) and aggressive periodontitis (also known as early onset periodontitis, destructive periodontitis, and juvenile periodontitis). Gingivitis treatment involves removing dental plaques and maintaining good oral hygiene. Periodontitis therapy should include root debriding, draining the infected root, and surgically resecting inflamed periodontal tissues. Systemic antimicrobials often are indicated in NUG, chronic periodontitis, and aggressive periodontitis. When possible, antimicrobial selection should be based upon culture and susceptibility testing of the subgingival flora.