E. Gemmell

Cytokines in periodontal disease: where to from here?

Numerous studies have attempted to elucidate the cytokine networks involved in chronic periodontitis, often with conflicting results. A variety of techniques were used to study cells in situ, cells extracted from gingival tissues, peripheral blood mononuclear cells, purified cell populations, and T cell lines and clones. Bacterial components, including sonicates, killed cells, outer membrane components, and purified antigens, have all been used to stimulate cells in vitro, making comparisons of cytokine profiles difficult. As it is likely that different cells are present at different disease stages, the inability to determine disease activity clinically is a major limitation of all these studies. In the context of tissue destruction, cytokines such as IL-1, IL-6 and IL-18 are likely to be important, as are their regulating cytokines IL-10 and IL-11. In terms of the nature of the inflammatory infiltrate, two apparently conflicting hypotheses have emerged: one based on direct observations of human lesions, the other based on animal experimentation and the inability to demonstrate IL-4 mRNA in gingival extracts. In the first of these, Th1 responses are responsible for the stable lesion, while in the second Th2 responses are considered protective. Using Porphyromonas gingivalis-specific T cell lines we have shown a tendency for IFN-γ production rather than IL-4 or IL-10 when antigen is presented with peripheral blood mononuclear cells which may contain dendritic cells. It is likely that the nature of the antigen-presenting cell is fundamental in determining the nature of the cytokine profile, which may in turn open up possibilities for new therapeutic modalities.

Cytokine Profiles of Cells Extracted from Humans with Periodontal Diseases

FACS analysis was used to determine the percent interferon (IFN)-gamma-, interleukin (IL)-4-, IL-10-, and CD30-positive T-cells extracted from adult periodontitis (AP) and healthy/gingivitis (H/G) subjects. Additionally, the percentages of IL-1β-, IL-10- and IL-12-producing B-cells and macrophages were ascertained. The percent IL-10+ CD8 cells extracted from AP lesions was decreased compared with H/G tissues (p = 0.033), and the percent CD30+ CD4 (p = 0.001) and CD30+ CD8 (p = 0.028) cells was higher in AP than in H/G tissues. The percentages of IL-1β+ macrophages (p = 0.003) and IL-12+ B-cells (p = 0.034) were both higher in AP lesions than in H/G tissues. The specific effect of Porphyromonas gingivalis on the cytokine profiles of peripheral blood mononuclear cells isolated from P. gingival is-infected AP and H/G patients was also determined. While there were no significant differences in the percent cytokine-positive T-cells after stimulation with P. gingival is outer membrane antigens (OM) for 6 days compared with cells incubated in medium only, the percent CD30+ CD4 cells increased significantly (p = 0.047 and p = 0.063 for AP and H/G groups, respectively). There was also an increase in the percent IL-1β+ B-cells from AP patients (p = 0.029), and the percent IL-12+ monocytes from AP and H/G subjects was higher than the percent IL-12+ B-cells, both after stimulation with P. gingivalis OM (p = 0.005 for the AP group and p = 0.058 and therefore not quite significant for the H/G group) and when incubated in medium alone (p = 0.016 and p = 0.015 for AP and H/G groups, respectively). This study has shown that IL-10+ CD8 cells may be significant in gingival lesions, and that CD30+ T-cells indicative of Th2 or Th0 cells may play a role in progressive periodontal disease. This study has also shown that B-cells produce IL-1 in the gingival lesion and that P. gingivalis may be significant in the induction of B-cell-induced IL-1.

Anti-P. gingivalis Response Correlates with Atherosclerosis

Significant associations between atherosclerosis and both Porphyromonas gingivalis, a major periodontopathogen, and the respiratory pathogen, Chlamydia pneumoniae, have been shown. Many individuals with evidence of atherosclerosis demonstrate seropositivity to these pathogens. The aim of the present study was to examine the atherogenic effect of repeated immunizations with either or both of these agents, and to determine if molecular mimicry of bacterial heat-shock protein (HSP), termed GroEL, and host (h) HSP60 was involved. Atherogenesis was examined in apolipoprotein-E-deficient (−/−) mice following intraperitoneal immunizations with P. gingivalis, C. pneumoniae, P. gingivalis, and C. pneumoniae or vehicle. Lesion area in the proximal aorta and levels of serum antibodies to P. gingivalis, C. pneumoniae, and GroEL were measured. The increased pathogen burden of P. gingivalis, but not of C. pneumoniae, enhanced atherosclerosis. hHSP60 was detected in lesions, and in P. gingivalis-immunized mice, lesion development was correlated with anti-GroEL antibody levels, supporting the involvement of molecular mimicry between GroEL and hHSP60.

Characterization of heat shock protein-specific T cells in atherosclerosis.

ABSTRACT A role for infection and inflammation in atherogenesis is widely accepted. Arterial endothelium has been shown to express heat shock protein 60 (HSP60) and, since human (hHSP60) and bacterial (GroEL) HSP60s are highly conserved, the immune response to bacteria may result in cross-reactivity, leading to endothelial damage and thus contribute to the pathogenesis of atherosclerosis. In this study, GroEL-specific T-cell lines from peripheral blood and GroEL-, hHSP60-, and Porphyromonas gingivalis-specific T-cell lines from atherosclerotic plaques were established and characterized in terms of their cross-reactive proliferative responses, cytokine and chemokine profiles, and T-cell receptor (TCR) Vβ expression by flow cytometry. The cross-reactivity of several lines was demonstrated. The cytokine profiles of the artery T-cell lines specific for GroEL, hHSP60, and P. gingivalis demonstrated Th2 phenotype predominance in the CD4 subset and Tc0 phenotype predominance in the CD8 subset. A higher proportion of CD4 cells were positive for interferon-inducible protein 10 and RANTES, with low percentages of cells positive for monocyte chemoattractant protein 1 and macrophage inflammatory protein 1α, whereas a high percentage of CD8 cells expressed all four chemokines. Finally, there was overexpression of the TCR Vβ5.2 family in all lines. These cytokine, chemokine, and Vβ profiles are similar to those demonstrated previously for P. gingivalis-specific lines established from periodontal disease patients. These results support the hypothesis that in some patients cross-reactivity of the immune response to bacterial HSPs, including those of periodontal pathogens, with arterial endothelial cells expressing hHSP60 may explain the apparent association between atherosclerosis and periodontal infection.

Physiological changes in brushtail possums, Trichosurus vulpecula, transferred from the wild to captivity

To determine the effect of relocation on the health of possums, the body weights and hormone and immune responses of 11 male and 9 female brushtail possums were monitored following transfer from the environs of Brisbane into an established breeding colony in Brisbane. The possums were monitored weekly for the first 20 weeks of captivity, and their immune responses assessed again 12 months after capture. Over the first 5 weeks of captivity, male possums lost a mean of 8.8% of their original body weight, and females lost 15.3% over the first 6 weeks. Variation between individual possums was evident, and the 11 male possums could be divided into two groups, those that gained weight (number of animals, N = 4) and those that lost weight (N = 7) in captivity. Four males gained weight following capture, and their body weight after 20 weeks of captivity was higher than at capture. The remaining seven males lost weight over the 20 weeks following introduction into captivity, resulting in a lower weight at week 20 than at capture. All of the nine female possums lost weight and were slower to regain weight compared to the males. Plasma cortisol concentrations did not vary greatly over the 20 weeks in male possums, and the mean plasma concentration of cortisol for the 11 male possums was 7.8 ng/ml (number of samples, n = 220). The female possums showed a different pattern. The concentration of cortisol for the nine female possums at week 1 was 34.0 ng/ml, which was significantly higher than 13.3 ng/ml at week 20 (P < 0.016). No significant variation in the mean concentration of plasma thyroxine of 5.7 ng/ml occurred in the 11 male possums over the 20-week period (n = 220). The plasma concentration of thyroxine for the nine female possums was 2.5 ng/ml (n = 54) for the first 6 weeks. At week 6, an increase in the concentration of thyroxine occurred, and a peak concentration of 6.9 ng/ml was reached at week 13. This increase correlated with the females regaining body weight. A low concentration of thyroxine is often associated with stress, thus an increase in the concentration of this hormone, combined with an increase in body weight, may indicate that these females had begun to adjust to their new environment. The seven male possums that lost weight following introduction into captivity displayed a significantly higher concentration of cortisol (9.1 compared with 5.3 ng/ml P < 0.01), and a lower concentration of thyroxine compared to the four males that gained weight following capture (4.7 compared with 7.3 ng/ml, P < 0.005). Over the 20-week period, the total number of white blood cells increased, and the number of neutrophils increased in both males and females. The proliferative response of lymphocytes from male possums to the T-cell mitogen, phytohaemagglutin (PHA) decreased significantly over the 20-week period (P < 0.002). In females an initial decrease in the reactivity of lymphocytes observed over the first 10 weeks was followed by an increase in this response over the remaining 10-week period. Twelve months following capture, the white blood cell parameters of both males and females had returned to similar levels to those of the first 1-5 weeks. The reactivity of lymphocytes from male possums that had been in captivity for 12 months was significantly higher than that of the first 20 weeks of captivity (P < 0.005). Females that had been in captivity for 12 months displayed lymphocyte responses similar to those observed at weeks 16-20. The body weight and hormonal results would suggest that possums undergo a more severe stress response than males immediately following their capture. In contrast, the immune response of males is lower than females and is depressed for a longer period following capture.

Mast Cells in Human Periodontal Disease

Recently, mast cells have been shown to produce cytokines which can direct the development of T-cell subsets. The aim of the present study was to determine the relationship between mast cells and the Th1/Th2 response in human periodontal disease. Tryptase+ mast cell numbers were decreased in chronic periodontitis tissues compared with healthy/gingivitis lesions. Lower numbers of c-kit+ cells, which remained constant regardless of clinical status, indicate that there may be no increased migration of mast cells into periodontal disease lesions. While there were no differences in IgG2+ or IgG4+ cell numbers in healthy/gingivitis samples, there was an increase in IgG4+ cells compared with IgG2+ cells in periodontitis lesions, numbers increasing with disease severity. This suggests a predominance of Th2 cells in periodontitis, although mast cells may not be the source of Th2-inducing cytokines.

Interleukin 18 and Periodontal Disease

Cytokines are of major importance in periodontal disease progression. It is generally agreed that control of the Th1/Th2 balance is central to the immunoregulation of periodontal disease. There is increasing evidence in humans that the stable periodontal lesion is mediated by Th1 cells, while the progressive lesion sees a shift toward Th2 cells. Equally, there is conflicting evidence, mainly in animal models, that bone loss is mediated by Th1 responses, and that Th2 responses are protective. In the presence of IL-12, IL-18 induces Th1 responses while, in the absence of IL-12, it promotes Th2 responses. It is clear, therefore, that since IL-18 has the ability to induce either Th1 or Th2 differentiation, it becomes important to consider its role in periodontal disease. This review endeavors to give an overview of this cytokine and its relevance for periodontal disease.

Cytokines and T Cell Switching

In recent years, the phenotypic characterization of T cell subsets has given way to a functional dichotomy based essentially on their cytokine profiles. In this context, the CD4+ helper T cell subset has been shown to consist of two types, termed Th1 and Th2. In general, Th1 cells produce interleukin (IL)-2 and interferon (IFN)-gamma, while Th2 cells characteristically produce IL-4, IL-5, and IL-6. The major function of the Th1 subset is to mediate delayed-type hypersensitivity reactions and their secondary function is suppression of B cell activity. In contrast, the major function of the Th2 subset is to provide B cell help, while their secondary function is cell-mediated immune suppression. A similar dichotomy has also been described for CD8+ T cells. The role that these functional T cell subsets and their cytokines play in terms of their protective and nonprotective outcomes in a variety of infectious and oral diseases is reviewed.

Effect of Fusobacterium nucleatum on the T and B cell responses to Porphyromonas gingivalis in a mouse model

T cell cytokine profiles and specific serum antibody levels in five groups of BALB/c mice immunized with saline alone, viable Fusobacterium nucleatum ATCC 25586, viable Porphyromonas gingivalis ATCC 33277, F. nucleatum followed by P. gingivalis and P. gingivalis followed by F. nucleatum were determined. Splenic CD4 and CD8 cells were examined for intracytoplasmic interleukin (IL)‐4, interferon (IFN)‐gamma and IL‐10 by dual colour flow cytometry and the levels of serum anti‐F. nucleatum and anti‐P. gingivalis antibodies determined by an ELISA. Both Th1 and Th2 responses were demonstrated by all groups, and while there were slightly lower percentages of cytokine positive T cells in mice injected with F. nucleatum alone compared with the other groups immunized with bacteria, F. nucleatum had no effect on the T cell production of cytokines induced by P. gingivalis in the two groups immunized with both organisms. However, the percentages of cytokine positive CD8 cells were generally significantly higher than those of the CD4 cells. Mice immunized with F. nucleatum alone had high levels of serum anti‐F. nucleatum antibodies with very low levels of P. gingivalis antibodies, whereas mice injected with P. gingivalis alone produced anti‐P. gingivalis antibodies predominantly. Although the levels of anti‐F. nucleatum antibodies in mice injected with F. nucleatum followed by P. gingivalis were the same as in mice immunized with F. nucleatum alone, antibody levels to P. gingivalis were very low. In contrast, mice injected with P. gingivalis followed by F. nucleatum produced equal levels of both anti‐P. gingivalis and anti‐F. nucleatum antibodies, although at lower levels than the other three groups immunized with bacteria, respectively. Anti‐Actinobacillus actinomycetemcomitans, Bacteroides forsythus and Prevotella intermedia serum antibody levels were also determined and found to be negligible. In conclusion, F. nucleatum immunization does not affect the splenic T cell cytokine response to P. gingivalis. However, F. nucleatum immunization prior to that of P. gingivalis almost completely inhibited the production of anti‐P. gingivalis antibodies while P. gingivalis injection before F. nucleatum demonstrated a partial inhibitory effect by P. gingivalis on antibody production to F. nucleatum. The significance of these results with respect to human periodontal disease is difficult to determine. However, they may explain in part differing responses to P. gingivalis in different individuals who may or may not have had prior exposure to F. nucleatum. Finally, the results suggested that P. gingivalis and F. nucleatum do not induce the production of cross‐reactive antibodies to other oral microorganisms.

T-cell antigen specificity in humans following stimulation with Porphyromonas gingivalis

The effects of Porphyromonas gingivalis stimulation on T-cell clonality and cytokine mRNA expression in peripheral blood mononuclear cells from individuals with gingivitis and periodontitis were investigated. Clonality of T cells was investigated by reverse transcription-polymerase chain reaction (RT-PCR) and single-strand conformation polymorphism analysis. Cytokine mRNA expression was investigated by RT-PCR. Whereas unstimulated mononuclear cells did not demonstrate obvious clonality, clonal expansion was found in most Vbeta families after stimulation. However, there was no relation between clonal change and disease category or the presence of P. gingivalis infection. Messenger RNA for interferon-gamma and interleukin-13 was upregulated whereas interleukin-4 and -10 were downregulated following P. gingivalis stimulation. Interleukin-12p35 demonstrated no consistent pattern. This study supports the concept that P. gingivalis stimulates T cells in an antigen-specific fashion. It further suggests that peripheral blood T cells may preferentially produce interferon-gamma and interleukin-13 in response to P. gingivalis stimulation irrespective of disease or P. gingivalis status.