ABSTRACT
BACKGROUND: T cells are fundamental in the pathogenesis of periodontal disease. Suppression of cell-mediated responses is associated with disease progression together with the concomitant increase in plaque pathogens including Porphyromonas gingivalis. The aim of the present study was to examine gene expression in T cells in response to P. gingivalis in mice. METHODS: BALB/c mice were given weekly intraperitoneal injections of P. gingivalis outer-membrane antigens with Freund's incomplete adjuvant for 3 weeks, whereas control mice received phosphate buffered saline (PBS) and adjuvant only. Splenic CD4 and CD8 subpopulations were isolated by magnetic cell separation and their responses investigated using microarray analysis. RESULTS: Most genes coded for enzymes concerned with metabolic pathways. Only five and 28 genes, respectively, were upregulated in CD4 and CD8 cells extracted from P. gingivalis-immunized mice, including immunoglobulin (Ig) heavy-chain genes for IgG1 and IgG2a in CD4 cells. In contrast, 1,141 and 1,175 genes, respectively, were downregulated. A total of 60 and 65 genes, respectively, coded for immune response proteins or those relevant to periodontal disease pathogenesis. The overlap of genes in the two subsets was 21%. One of the major effects, apart from T-cell function suppression, was the shift away from Th1 responses, although there was also a downregulation of two genes and upregulation of one Th2-response gene. Genes downregulated included those encoding cytokines, proteins involved in Ig binding, antigen presentation, innate immunity, extracellular matrix, and cell adhesion molecules that could result in dysregulation in the progressive periodontal lesion. CONCLUSIONS: Early findings in humans demonstrated that periodontopathic bacteria induce immunosuppressive effects on T cells. The present study has shown that P. gingivalis had a predominant downregulatory effect on gene expression in CD4 and CD8 T cells in mice.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Gene Expression/immunology , Immunocompetence/genetics , Periodontitis/immunology , Periodontitis/microbiology , Porphyromonas gingivalis/immunology , Animals , Bacterial Outer Membrane Proteins/immunology , CD4-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/metabolism , Down-Regulation/immunology , Female , Immunoproteins/biosynthesis , Mice , Mice, Inbred BALB C , Porphyromonas gingivalis/pathogenicity , Protein Array Analysis , Spleen/cytologyABSTRACT
BACKGROUND: It has previously been suggested that CD4+ T cells play a pivotal role in regulating the immune response to periodontal pathogens. The aim of the present study therefore was to determine delayed type hypersensitivity (DTH), spleen cell proliferation, serum and splenic anti-Porphyromonas gingivalis antibody levels, and lesion sizes following challenge with viable P. gingivalis in CD4-depleted BALB/c mice immunized with P. gingivalis outer membrane proteins (OMP). METHODS: Four groups of BALB/c mice were used. Groups 1 and 2 were injected intraperitoneally (ip) with saline for 3 consecutive days and then weekly throughout the experiment. Groups 3 and 4 were injected ip with rat immunoglobulin and a monoclonal rat anti-mouse CD4 antibody, respectively. Two days later, group 1 mice were injected ip with saline only, while all the other groups were immunized ip with P gingivalis OMP weekly for 3 weeks. One week later following the last immunization of OMP, 3 separate experiments were conducted to determine: 1) the DTH response to P gingivalis OMP by measuring footpad swelling; 2) the levels of antibodies to P gingivalis in serum samples and spleen cell cultures using an enzyme-linked immunosorbent assay, as well as spleen cell proliferation after stimulation with OMP; and 3) the lesion sizes after a subcutaneous challenge with viable P. gingivalis cells. RESULTS: In CD4+ T-cell-depleted mice (group 4), the DTH response and antigen-stimulated cell proliferation were significantly suppressed when compared to groups 2 and 3. Similarly, the levels of serum and splenic IgM, IgG, and all IgG subclass antibodies to P. gingivalis OMP were depressed. Delayed healing of P gingivalis-induced lesions was also observed in the CD4+ T-cell-depleted group. CONCLUSIONS: This study has shown that depletion of CD4+ T cells prior to immunization with P gingivalis OMP led to the suppression of both the humoral and cell-mediated immune response to this microorganism and that this was associated with delayed healing. These results suggest that the induction of the immune response to P. gingivalis is a CD4+ T-cell-dependent mechanism and that CD4+ T cells are important in the healing process.
Subject(s)
CD4-Positive T-Lymphocytes/physiology , Porphyromonas gingivalis/immunology , Abscess/immunology , Abscess/microbiology , Animals , Antibodies, Bacterial/biosynthesis , Bacterial Outer Membrane Proteins/immunology , Enzyme-Linked Immunosorbent Assay , Female , Foot , Hypersensitivity, Delayed , Immunization , Mice , Mice, Inbred BALB C , Porphyromonas gingivalis/pathogenicity , Spleen/cytology , Spleen/immunology , Wound Healing/immunologyABSTRACT
BACKGROUND: Susceptibility to periodontal infections may, in part, be genetically determined. Porphyromonas gingivalis is a major periodontopathogen, and the immune response to this organism requires T-cell help. The aim of the present study was to examine the specific T-cell cytokine responses to P. gingivalis outer membrane antigens in a mouse model and their relationship with H-2 haplotype. METHODS: BALB/c and DBA/2J (H-2d), CBACaH (H-2k), and C57BL6 (H-2b) mice were immunized with P. gingivalis outer membrane antigens weekly for 3 weeks. One week after the final injection, the spleens were removed, and 6 T-cell lines specific for P. gingivalis were established for each mouse strain. The percentage of CD4 and CD8 cells in the P. gingivalis-specific T-cell lines staining positive for intracytoplasmic interleukin (IL)-4, interferon (IFN)-gamma, and IL-10 was determined by 2-color flow cytometry. RESULTS: The cytokine profiles of T-cell lines from BALB/c and DBA/2J mice showed no significant differences. Significantly fewer IL-4+, IFN-gamma+, and IL-10+ CD4 cells than IL-4+, IFN-gamma+, and IL-10+ CD8 cells, respectively, were demonstrated for both strains. P. gingivalis-specific T-cell lines generated from CBACaH mice were similar to those generated from BALB/c and DBA/2J mice; however, the mean percentage of IL-4+ CD4 cells in CBACaH mice was lower than the percentage of IFN-gamma+ CD4 cells. Also, the mean percentage of IFN-gamma+ CD4 cells in CBACaH mice was significantly increased compared to DBA/2J mice. Unlike the other 3 strains, T-cell lines established from C57BL6 mice contained similar percentages of cytokine-positive cells, although the percentage of IL-4+ CD4 cells was reduced in comparison to the percentage of CD8 cells. However, comparisons with the other 3 strains demonstrated a higher percentage of IL-4+ CD4 cells than in lines established from the spleens of DBA/2J mice, IFN-gamma+ CD4 cells than in lines established from BALB/c and CBACaH mice, and IL-10+ CD4 cells than in lines established from all 3 other strains. No significant differences in the percentage of positive CD8 cells were demonstrated between lines in the 4 strains of mice. CONCLUSION: The specific T-cell response to P. gingivalis in mice may, in the case of the CD4 response, depend on MHC genes. These findings are consistent with the concept that patient susceptibility is important to the outcome of periodontal infection and may, in part, be genetically determined.
Subject(s)
CD4-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/metabolism , Cytokines/biosynthesis , Cytokines/genetics , Porphyromonas gingivalis/immunology , T-Lymphocyte Subsets/metabolism , Animals , Antibodies, Bacterial/biosynthesis , Antigens, Bacterial , Bacterial Outer Membrane Proteins/immunology , Cell Line/immunology , Female , Flow Cytometry , H-2 Antigens/genetics , Immunization , Interferon-gamma/biosynthesis , Interferon-gamma/genetics , Interleukins/biosynthesis , Interleukins/genetics , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Inbred CBA , Mice, Inbred DBAABSTRACT
OBJECTIVES: The aim of this study was to determine the nature of the inflammatory infiltrate associated with different transmucosal implant surfaces in dogs. METHODS: Three experimental and one control single-stage implants were randomly placed on each side of the jaw in eight dogs. The transmucosal portion of the test implants consisted of an acid-etched surface (type A), a machined surface with a circumferential groove (type C) and a surface prepared by mild anodic oxidation (type D). The control was a standard machined surface (type B). In order to determine the response to the different surfaces, plaque control was carried out twice weekly following placement of the implants for the entire period of the experiment. At 6 months, gingival biopsies and plaque samples were obtained. The area of inflammatory infiltrate and the nature of the infiltrating cell types were determined using immunohistology. Real-time polymerase chain reaction was used to identify putative periodontal pathogens. RESULTS: Inflammatory infiltrates were associated with all implant surfaces and were commonly found subepithelially and perivascularly. T cells were the predominant infiltrating cell type in all lesions, associated with the different surfaces. In all lesions the CD4 : CD8 ratio was approximately 2 : 1. Statistical analysis showed that the type C surface (machined surface with a groove) had significantly larger inflammatory infiltrates than the type B surface (machined surface without a groove; P<0.05). No statistically significant differences were found with respect to the size of the inflammatory infiltrates or in terms of the nature of infiltrating cells. However, despite the intensive plaque control regime, plaque was present on all implant surfaces at the time of biopsy 6 months after placement. All implants had similar numbers of Tannerella forsythia, Fusobacterium nucleatum and Porphyromonas gingivalis. Actinobacillus actinomycetemcomitans, was not detected in any sample. CONCLUSIONS: These results suggest that the development of inflammation associated with implants is independent of surface type, but is nevertheless associated with the presence of plaque. The different surfaces had no influence on the nature of the infiltrate, with T cells being the predominant cell type in all lesions. Finally, the different implant surface types seemed not to influence the peri-implant microbiota. However, the presence of the circumferential groove tended to be associated with larger infiltrates. Whether this is due to increased plaque accumulation remains to be determined.
Subject(s)
Dental Implants , Dental Prosthesis Design , Periodontitis/etiology , Acid Etching, Dental/methods , Animals , B-Lymphocytes/pathology , Bacteria/classification , Bacteroides/classification , CD4-CD8 Ratio , Dental Implants/adverse effects , Dental Implants/microbiology , Dental Plaque/etiology , Dental Plaque/microbiology , Dental Prosthesis Design/adverse effects , Dogs , Epithelial Attachment/pathology , Epithelium/pathology , Fusobacterium nucleatum/isolation & purification , Gingiva/pathology , Oxidation-Reduction , Periodontitis/microbiology , Porphyromonas gingivalis/isolation & purification , Random Allocation , Surface Properties , T-Lymphocytes/pathologyABSTRACT
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) Vbeta 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 1alpha, whereas a high percentage of CD8 cells expressed all four chemokines. Finally, there was overexpression of the TCR Vbeta5.2 family in all lines. These cytokine, chemokine, and Vbeta 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.