D. F. Kinane

Cytokine Responses of Oral Epithelial Cells to Porphyromonas gingivalis Infection

Accumulating evidence indicates that epithelia are not merely mechanical barriers but also important elements of the innate immune system. The present study was performed to examine cytokine responses of oral epithelial cells after infection with the periodontal pathogen Porphyromonas gingivalis. The KB-cell line and primary cultures of periodontal pocket epithelium were infected with P. gingivalis for assessment of bacterial invasion by an antibiotic protection assay, and examinination of expression of interleukin-1 beta, interleukin-6, interleukin-8, and tumor necrosis factor-alpha by in situ hybridization and immunohistochemistry. We observed that P. gingivalis induces a strong cytokine response, positively correlated with the adhesive/invasive potential of the infecting strain, in both KB cells and primary cultures. These findings indicate that the epithelial cells of the periodontal pocket are an integral part of the immune system, eliciting cytokine responses to a bacterial challenge. In this context, the adhesive/invasive phenotype of P. gingivalis appears to contribute to pathogenicity.

Scientific Basis of a Matrix Metalloproteinase‐8 Specific Chair‐side Test for Monitoring Periodontal and Peri‐implant Health and Disease

ABSTRACT: Matrix metalloproteinases (MMPs), especially collagenase‐2 (MMP‐8), are key mediators of irreversible tissue destruction associated with periodontitis and peri‐implantitis. MMP‐8 is known to exist in elevated amounts and in active form in the gingival crevicular fluid (GCF) and peri‐im‐plant sulcular fluid (PISF) from progressing periodontitis and peri‐implantitis lesions and sites, respectively. (Sorsa et al. Ann. N.Y. Acad. Sci. 737: 112‐131 [1994]; Teronen et al. J. Dent. Res. 76: 1529‐1537 [1997]). We have developed monoclonal antibodies to MMP‐8 (Hanemaaijer et al. J. Biol. Chem. 272: 31504‐31509 [1997]) that can be used in a chair‐side dipstick test to monitor the course and treatment of periodontitis and peri‐implantitis. Monoclonal and polyclonal antibody tests for MMP‐8 coincided with the classical functional collagenase activity test from GCF and PISF (Sorsa et al. J. Periodont.Res. 22: 386‐393 [1988]) in periodontal and peri‐implant health and disease. In future a chair‐side functional and/or immunological MMP‐test can be useful to diagnose and monitor periodontal and peri‐implant disease and health.

Anti-inflammatory cytokine IL-10 and T cell cytokine profile in periodontitis granulation tissue

Th2 cells are more abundant than Th1 cells in periodontitis lesions, but the relative importance of the Th1 and Th2 subsets in periodontal disease is not understood. In addition, the role of proinflammatory and anti‐inflammatory cytokines in this disease process is unclear. Biopsies were obtained from 10 patients with early onset periodontitis (EOP) and 10 patients with adult periodontitis (AP). From all of the patients in the AP group we were able to obtain and section the gingival tissue to serve as controls. We used polyclonal monospecific antibodies to detect cells expressing IL‐2, IL‐4, IL‐6, IL‐10 and IL‐15, tumour necrosis factor (TNF‐α) and interferon‐gamma (IFN‐γ) in formalin‐fixed, paraffin‐embedded sections of granulation tissue from periodontitis lesions. We also employed a series of oligonucleotide probes to detect cells expressing the cytokine transcripts in the same tissue biopsies. Cells that expressed IL‐4 or IL‐6 were more numerous than cells expressing either IL‐2 or IFN‐γ. Th2 cells were more numerous in EOP and AP tissues. IL‐15 substitutes for IL‐2 in a number of biological activities related to the Th1 immune response, and interestingly, in periodontal lesions the IL‐15‐expressing cells outnumbered IL‐2‐expressing cells, suggesting that this is the pattern of immune regulation by T cells in the periodontium. The functional balance in the T cell subsets detected by their cytokine profiles underlies the importance of the anti‐inflammatory mechanisms taking place in the diseased tissue. The numbers of inflammatory leucocytes that express the anti‐inflammatory cytokine IL‐10 are much more widely distributed than those that express the proinflammatory cytokines IL‐6 and TNF‐α. This study suggests that large numbers of infiltrating inflammatory cells as well as accessory cells are involved in the down‐regulation of the inflammatory and immune response in periodontitis.

Clinical, pathological and immunological aspects of periodontal disease

The inflammatory and immune responses during the development and progression of periodontitis are reviewed. Susceptibility to periodontitis may be related to whether plasma cells predominate in the tissues of an individual, or a site, in response to the microbial insult from dental plaque. The tendency for an individual or site to form an extensive plasma cell infiltrate may indicate an inability to defend against periodontopathogenic bacteria and thus a predisposition to periodontitis. Selected pertinent areas of current interest in cellular and humoral immunology are considered within the periodontal context. These topical issues include (a) homing of immune and inflammatory cells to target tissues; (b) the local proliferation and synthetic activity of immune and inflammatory cells; (c) the cytokine profile of the inflammatory and immune cells; and (d) the immunoglobulin subclasses of locally produced antibodies.

An evaluation of the Nd:YAG laser in periodontal pocket therapy.

The aim of this study was to determine whether the Nd:YAG laser energies of 50 and 80 mJ at 10 pulses per second (pps) were capable of improving the clinical parameters associated with periodontal disease. These energy settings were chosen as previous work indicated that higher values would damage root surfaces and that 80 mJ had an in-vitro bactericidal effect. Eighty periodontally affected sites in teeth scheduled for extraction from 11 patients with adult periodontitis were randomly placed in one of the following four treatment groups: 1. laser treatment at 50 mJ, 10 pps for 3 minutes; 2. laser treatment at 80 mJ, 10 pps for 3 minutes; 3. scaling and 4. untreated control. Probing depth, bleeding on probing (BOP), plaque index, gingival index and gingival crevicular fluid (GCF) volume were measured at baseline and week 6. Baseline subgingival microbiological samples were collected, then repeated immediately after treatment and at week 6 to assess the total anaerobic colony forming units (CFU). Only the scaling group showed a significant reduction in pocket depth and BOP (P < 0.001). The microbial samples taken immediately after scaling and laser at 80 mJ and 10 pps treatments showed a significant reduction in total CFU compared with the baseline (P < 0.01), which was sustained only in the scaling group until week 6. Electron microscopy did not reveal any heat damage on the root surfaces. This study demonstrated that application of Nd:YAG laser pulses of 50 mJ and 80 mJ failed to improve the clinical and microbiological parameters of periodontal disease

Bioassay of interleukin 1 (IL-1) in human gingival crevicular fluid during experimental gingivitis

The cytokine IL-1 was demonstrated in crevicular fluid during a 14- and 21-day experimental gingivitis in healthy human volunteers. A sensitive and specific bioassay allowed detection of biologically active IL-1 at levels ranging from 0.18 ng/microliters at baseline to 1.70 ng/microliters in inflamed gingiva. Levels of IL-1 increased rapidly with plaque accumulation and in advance of the subsequent gingival inflammation, peaking within 7 days of the start of gingivitis. As changes in IL-1 were detected before clinically recognizable gingival changes, IL-1 may have potential as an early marker of gingival inflammatory changes.

Humoral immune responses in periodontal disease may have mucosal and systemic immune features

The humoral immune response, especially IgG and IgA, is considered to be protective in the pathogenesis of periodontal disease, but the precise mechanisms are still unknown. Immunoglobulins arriving at the periodontal lesion are from both systemic and local tissue sources. In order to understand better the local immunoglobulin production, we examined biopsy tissue from periodontitis lesions for the expression of IgM, IgG, IgA, IgE and in addition the IgG and IgA subclasses and J‐chain by in situ hybridization. Tissues examined were superficial inflamed gingiva and the deeper granulation tissue from periodontal sites. These data confirm that IgM, and IgG and IgA subclass proteins and J‐chain can be locally produced in the periodontitis tissues. IgG1 mRNA‐expressing cells were predominant in the granulation tissues and in the gingiva, constituting approx. 65% of the total IgG‐expressing plasma cells. There was a significantly increased proportion of IgA‐expressing plasma cells in the gingiva compared with the granulation tissue (P < 0.01). Most of the IgA‐expressing plasma cells were IgA1, but a greater proportion expressed IgA2 mRNA and J‐chain mRNA in the gingival tissues (30.5% and 7.5%, respectively) than in the periodontal granulation tissues (19% and 0–4%, respectively). The J‐chain or dimeric IgA2‐expressing plasma cells were located adjacent to the epithelial cells, suggesting that this tissue demonstrates features consistent with a mucosal immune response. Furthermore, we were able to detect the secretory component in gingival and junctional epithelial cells, demonstrating that the periodontal epithelium shares features with mucosal epithelium. In contrast, deeper tissues had more plasma cells that expressed IgM, and less expressing IgA, a response which appears more akin to the systemic immune response. In conclusion, this study suggests that immune mechanisms involved in the pathogenesis of periodontitis may involve features of both the mucosal and systemic immune systems, dependent on tissue location.

Clinical and Genetic Analysis of a Large North European Caucasian Family Affected by Early-onset Periodontitis

Genetic studies of early-onset periodontitis (EOP) are hampered by several factors. These include delayed onset of the trait, an upper age limit of expression of the disease, and lack of phenotypic information for edentulous family members. Segregation analyses of families with EOP support a major locus hypothesis but fail to define clearly the criteria used for diagnosis of the relatives. Confirmation of a proposed mode of inheritance and the identification of risk genes is awaited by means of family linkage studies. It is suggested that a system can be developed for the current and retrospective diagnosis of relatives of EOP probands. In addition, it is hypothesized that the large family presented here is suitable for a linkage study. Relatives of the proband who were unavailable for a full periodontal examination, were edentulous, or were deceased, were diagnosed by means of documented clinical evidence of periodontal disease or from reported case histories. Segregation analysis was performed. Analysis of the power of the pedigree to detect linkage was carried out by means of the SIMLINK program. Three different categories were defined according to the reliability of diagnosis of EOP. Segregation analysis indicated either autosomal-dominant or X-linked-dominant inheritance in this family. The simulations showed lod scores above 3.0 for all locations of the disease gene, and for each category of diagnosis. In conclusion, a method has been developed which can be used for the diagnosis of relatives of EOP probands when ideal clinical data are unavailable. The simulations suggest that this family is suitable for a genetic linkage study with the aim of identifying the location of one or more susceptibility genes.

IgG and IgA subclass mRNA-bearing plasma cells in periodontitis gingival tissue and immunoglobulin levels in the gingival crevicular fluid

The humoral immune response, especially the production of IgG and IgA, is considered to have a protective role in the pathogenesis of periodontal disease, but the precise mechanisms are still unknown. In order to determine local IgG and IgA production, we investigated the presence of human IgG and IgA subclass mRNA‐bearing plasma cells within periodontal tissue by in situ hybridization using digoxigenin‐labelled oligonucleotide probes in 24 gingival biopsy samples (pocket depth>5 mm) which were obtained from eight patients with adult periodontitis. Furthermore, we examined IgG and IgA subclass proteins and digested IgA1 Fab portions in the gingival crevicular fluid (GCF), corresponding to the sites from which the tissues were taken, by ELISA. IgG and IgA subclass mRNA‐expressing cells were detected in all serial formalin‐fixed/paraffin‐embedded gingival tissue sections sampled. Plasma cells showed strong cytoplasmic staining with a high contrast and a good retention of morphology with these probes. IgG1 mRNA‐expressing cells were predominant (mean 63%) and IgG2 mRNA‐expressing cells were present at around 23% of total IgG plasma cells, while IgG3 and IgG4 mRNA‐expressing cells were present to a much lesser extent (3% and 10%, respectively). Similar proportions of IgG subclass proteins in GCF were detected, which were also consistent with ‘normal’ serum levels. In terms of IgA subclass, IgA1 mRNA‐positive cells were predominant (mean 65.1%, P<0.001). In contrast, IgA2 protein in the GCF samples were detected at higher concentrations than IgA1 (P<0.001). The ratio of total IgG to IgA mRNA‐positive plasma cells was ≈7.5:1. There was a good correlation between the amounts of IgG subclass proteins in GCF and the number of IgG subclass mRNA‐positive cells in the same sites, but not between IgA subclass proteins and the number of IgA subclass mRNA‐positive cells. These data suggest that IgG and IgA subclass proteins can be locally produced in the periodontitis gingiva. In addition, as we detected IgA1 Fab fragments in GCF, this is further confirmation that secreted IgA1 protein in GCF may be digested by periodontal bacteria.

Detection of interleukin-1β mRNA-expressing cells in human gingival crevicular fluid by in situ hybridization

Interleukin-1 beta (IL-1 beta) mRNA-expressing cells in human gingival crevicular washings (GCW) obtained from patients with periodontitis and healthy controls were examined by in situ hybridization. GCW was done at 15 diseased sites [Gingival Index > 1; pocket depth > or = 5 mm] from five patients with adult periodontitis and eight clinically periodontal healthy sites from three volunteers GI < or = 1; PD < or = 3 mm), and then the samples were cytocentrifuged. In situ hybridization using digoxigenin-labelled oligonucleotide probe complementary to human IL-1 beta mRNA showed IL-1 transcripts in both polymorphonuclear leucocytes and mononuclear cells but not in epithelial cells in all GCW samples from diseased and healthy sites. Polymorphs were the predominant leucocytes in diseased and healthy sites, averaging 91.7 +/- 4.6 and 77.0 +/- 10.3%, respectively. The percentages of IL-1 beta mRNA-positive polymorphonuclear leucocytes in GCW samples from diseased and healthy sites were 92.3 +/- 4.7 and 80.9 +/- 10.3%, respectively. The IL-1 beta gene signals in individual cells were quantified in five samples (two healthy and three diseased sites). The mean amounts of IL-1 beta mRNA expression in polymorphonuclear leucocytes was higher than that of mononuclear cells in all samples and there was heterogeneity within the populations of polymorphonuclear and mononuclears cells in their ability to express the IL-1 beta gene. These findings indicate that IL-1 beta may be predominantly produced by polymorphonuclear leucocytes in the gingival crevice of patients with adult periodontitis and periodontally healthy controls.