Kohji Hara

Elevated humoral immune response to heat shock protein 60 (hsp60) family in periodontitis patients

The presence of antibodies to the 60‐kD human and Porphyromonas gingivalis GroEL hsp60 in the sera and inflamed gingival tissues of periodontitis patients was examined. In order to obtain the antigens, recombinant plasmids carrying human hsp60 and P. gingivalis GroEL genes were constructed and expressed as histidine‐tagged recombinant proteins. Immunoreactivities of these proteins were confirmed by MoAbs specific to mammalian hsp60 and cross‐reactive with both mammalian and bacterial hsp60. Western blot analysis clearly demonstrated that the number of periodontitis patients showing a positive response to P. gingivalis GroEL was higher than the number of periodontally healthy subjects. Furthermore, anti‐P. gingivalis GroEL antibody was detected in all samples of gingival tissue extracts. For human hsp60, a higher frequency of seropositivity was found in the periodontitis patients than in the healthy subjects. In addition, the periodontitis patients demonstrated stronger reactivity compared with the healthy subjects. Quantitative analysis of serum antibodies by ELISA also demonstrated that the levels of antibodies in the sera of patients were significantly higher than those of control subjects. In the gingival tissue extracts, seven out of 10 patients demonstrated a positive response to human hsp60 and tso of these demonstrated strong positivity. Affinity‐purified serum antibodies to human hsp60 and P. gingivalis GroEL from selected patients reacted with P. gingivalis GroEL and human hsp60, respectively, suggesting cross‐reactivity of antibodies. These results suggest that molecular mimicry between GroEL of the periodontopathic bacterium P. gingivalis and autologous human hsp60 may play some role in immune mechanisms in periodontitis.

Expression of mRNA for matrix metalloproteinases and tissue inhibitors of metalloproteinases in periodontitis-affected human gingival tissue

It is known that the host responds to an increased concentration of collagenase [or matrix metalloproteinase (MMP)-1] by preferentially expressing mRNA for the tissue inhibitor of metalloproteinase-1 (TIMP-1) in order to overcome tissue destruction due to periodontitis. To further elucidate the relation between MMPs and TIMPs in periodontitis-affected tissues, the expression of mRNA for MMP-1, -3 and -8, and TIMP-1 and -2, in 10 gingival samples from patients and five from healthy individuals was assessed by reverse transcription-polymerase chain reaction. The diseased group showed significantly higher levels of MMP-1, -3, -8 and TIMP-1 mRNA relative to beta-actin than the control group (mean +/- SE: diseased vs healthy (%): 0.26 +/- 0.05 vs 0.018 +/- 0.0040 for MMP-1; 0.09 +/- 0.16 vs 0.063 +/- 0.016 for MMP-3; 0.068 +/- 0.017 vs 0.006 +/- 0.0010 for MMP-8; 12.66 +/- 2.90 vs 2.71 +/- 0.54 for TIMP-1; p < 0.01). TIMP-2 did not significantly differ between the two groups (1.79 +/- 0.33 vs 1.42 +/- 0.53; p > 0.05). The preferential increase in the level of MMP-3 mRNA relative to that of MMP-1 and -8 in inflamed gingiva would be relevant to tissue destruction because MMP-3 is a broad-spectrum MMP and a pivotal activator of latent MMP-1 and -8. Therefore, the overall increase in MMP-1, -3 and -8 mRNA in periodontitis-affected gingiva might account for a concerted action of MMPs during connective tissue destruction in periodontitis.

A cell line with characteristics of the periodontal ligament fibroblasts is negatively regulated for mineralization and Runx2/Cbfa1/Osf2 activity, part of which can be overcome by bone morphogenetic protein-2.

The periodontal ligament (PDL) is a connective tissue located between the cementum of teeth and the alveolar bone of the mandibula. It plays an integral role in the maintenance and regeneration of periodontal tissue. The cells responsible for maintaining this tissue are thought to be fibroblasts, which can be either multipotent or composed of heterogenous cell populations. However, as no established cell lines from the PDL are available, it is difficult to assess what type of cell promotes all of these functions. As a first step to circumvent this problem, we have cloned and characterized cell lines from the PDL from mice harboring a temperature-sensitive SV 40 large T-antigen gene. RT-PCR and in situ hybridization studies demonstrated that a cell line, designated PDL-L2, mimics the gene expression of the PDL in vivo: it expresses genes such as alkaline phosphatase, type I collagen, periostin, runt-related transcription factor-2 (Runx2) and EGF receptor, but does not express genes such as bone sialoprotein and osteocalcin. Unlike osteoblastic cells and a mixed cell population from the PDL, PDL-L2 cells do not produce mineralized nodules in the minearlization medium. When PDL-L2 cells were incubated in the presence of recombinant human bone morphogenetic protein-2 alkaline phosphatase activity increased and mineralized nodules were eventually produced, although the extent of mineralization is much less than that in osteoblastic MC3T3-E1 cells. Furthermore, PDL-L2 cells appeared to have a regulatory mechanism by which the function of Runx2 is normally suppressed.

Relevance of local Th2-type cytokine mRNA expression in immunocompetent infiltrates in inflamed gingival tissue to periodontal diseases

It has been suggested that the types of inflammatory round cell infiltrates and the divergence in the cytokine production profile by macrophages and helper T cells regulate the course of infectious or inflammatory diseases, including periodontitis and gingivitis. We examined the expression of IL‐1α, IL‐1β, IL‐2, IL‐4, IL‐5, IL‐6 and tumour necrosis factor‐alpha (TNF‐α) mRNA in the inflamed gingiva by in situ hybridization. The results of single‐cell analysis were used as data sets for statistical analyses. The density of cells expressing IL‐1α, IL‐4 and IL‐5 mRNA was higher in periodontitis than in gingivitis. IL‐2 mRNA‐expressing cells were almost absent in gingivitis specimens. Principal component analysis disclosed three factors explaining 84.8% of the variance: one accounting for 40.5% of the variance and mainly regulated by IL‐1α, IL‐1β, IL‐6 and TNF‐α, and two others, explaining 29.9% and 14.4% of the variance, describing the relationship between the types of cytokines derived from macrophages or Th2 type. These results suggest that the cytokines produced by inflammatory cells infiltrating in the gingival tissue are influential on the progression of gingivitis, an acute and reversible inflammatory condition, to chronic and destructive periodontitis. Thus, periodontal disease progression may be regulated by the local cytokine network, and the bias in this network towards a Th2‐type cytokine dominance could be an exacerbating factor.

Immunohistological analysis of T cell functional subsets in chronic inflammatory periodontal disease.

IL‐2, interferon‐gamma (IFN‐γ), IL‐4 and IL‐6 producing T cells in periodontitis and gingivitis‐affected human tissues were investigated by immunohistochemistry to clarify the relationship between T cell functional subsets and disease entity. Using alkaline‐phosphatase anti‐alkaline‐phosphatase technique, the relative proportions of each cytokine‐producing T cell were calculated in the crevicular 1/3, middle 1/3 and oral 1/3 areas selected in the connective tissue of sections. CD19:CD3 and CD4:CD8 ratios were determined on the serial sections. Compared with gingivitis tissues, the proportion of cytokine‐producing cells in periodontitis‐affected samples was higher overall in the crevicular 1/3 (P < 0·02). The middle 1/3 exhibited a higher percentage of cytokine‐producing cells, except for IL‐6‐producing cells. Frequencies of cytokine‐producing cells in the oral 1/3 did not differ. IL‐4 was the prominent cytokine in periodontitis‐affected tissues, with the highest proportion detected in the crevicular 1/3. The CD19:CD3 ratio was higher in periodontitis tissues irrespective of the location, indicating a B cell dominance in periodontitis lesions. Furthermore, a significant positive correlation between the proportion of IL‐4‐producing cells and the CD19:CD3 ratio was noted. The CD4:CD8 ratio consistently exceeded 2·0 in both periodontitis and gingivitis. These results suggest that immunoregulation of both periodontitis and gingivitis are T cell‐dependent, but in periodontitis type 2 helper T cells predominate and thereby control B cell activation.

Elevated CTLA-4 expression on CD4 T cells from periodontitis patients stimulated with Porphyromonas gingivalis outer membrane antigen

To characterize the T cell response to Porphyromonas gingivalis, we examined the expression of costimulatory molecules on T cells derived from adult periodontitis patients with high serum antibody titre to P. gingivalis. The expression of CD28, CTLA‐4, CD40 ligand (CD40L) on CD4+ T cells was analysed by flow cytometry. IL‐10 and transforming growth factor‐beta (TGF‐β) mRNA expression were determined by reverse transcription‐polymerase chain reaction (RT‐PCR) and subsequent image analysis. Peripheral blood mononuclear cells (PBMC) derived from periodontitis patients showed higher proliferative responses to P. gingivalis outer membrane (OM) than those from healthy controls (P < 0.05). The percentage of CTLA‐4+ cells within CD4+ T cells of patients was significantly higher than that of healthy controls after P. gingivalis OM stimulation (33.0% versus 11.9%, P < 0.01). There was no significant difference in the percentages of CD28+ cells and CD40L+ cells, and the percentage of CD40L+ cells was low in both groups even after stimulation. Stimulation of PBMC with P. gingivalis OM induced significantly higher IL‐10 mRNA expression in periodontitis patients than in healthy controls (P < 0.05). The level of TGF‐β mRNA expression of patients tended to be higher than that of healthy controls, but there was no significant difference. To elucidate the functional role of CTLA‐4, we further investigated the secondary proliferative response to P. gingivalis OM. Interestingly, P. gingivalis OM stimulation did not enhance antigen‐specific secondary response. Anti‐CTLA‐4 MoAb had no effect on proliferation in the presence of P. gingivalis OM. CTLA‐4Ig suppressed the proliferative response significantly (P < 0.01). These results suggest that T cell responses to P. gingivalis OM may be regulated by CTLA‐4 that is expressed at the late phase of T cell activation, and, in part, immunosuppressive cytokines. Taken together, CTLA‐4 may play a crucial role in the pathogenesis of chronic inflammatory periodontal disease.

Differential expression of costimulatory molecules in chronic inflammatory periodontal disease tissue

Although B cell activation and subsequent immunoglobulin production are the immunopathological features of chronic inflammatory periodontal disease, in situ expression of costimulatory molecules in humoral immunity has not been investigated. In the present study we examined the expression of CD40, CD40 ligand (CD40L), CD80, CD86, CD28 and cytolytic T lymphocyte‐associated antigen‐4 (CTLA‐4) on lymphocytes immunohistochemically. Cryostat sections were prepared from the gingival tissue samples of 14 patients with moderate to advanced adult periodontitis. In vitro kinetics of the expression of CD40L and CTLA‐4 by peripheral blood T cells and that of CD80 and CD86 by peripheral blood B cells were also investigated by flow cytometry. Positive percentage expression of CD40L, CD28 and CTLA‐4, and CD40, CD80 and CD86 was calculated for the number of CD3+ and CD19+ cells, respectively. Flow cytometric analysis demonstrated that the expression of CD40L and CTLA‐4 on T cells, and CD80 and CD86 on B cells of peripheral blood was up‐regulated upon activation. While most T cells and B cells expressed CD28, and CD80 and CD86, respectively, in gingival tissues, the expression of CD40L and CTLA‐4 was lower but highly variable between specimens. Furthermore, these two molecules seemed to be expressed reciprocally in the lesion. As both CD40L and CTLA‐4 expression are induced transiently by stimulation, variability in the expression of the molecules may reflect immunological activities and participation in the regulation of B cell activation of the lesion.

ACTIVATION OF TRANSCRIPTION FACTORS AND IL-8 EXPRESSION IN NEUTROPHILS STIMULATED WITH LIPOPOLYSACCHARIDE FROM PORPHYROMONAS GINGIVALIS

DNA binding activity of NF-κB and AP-1 were examined in neutrophils stimulated with LPS purified from P. gingivalis, a major pathogenic bacteria of periodontitis lesion. Porphyromonas gingivalis LPS enhanced the activity reaching a peak at a concentration of 500 ng/ml in the absence of serum. The NF-κB activation stimulated with 10 ng/ml of P. gingivalis LPS was suppressed approximately 44% by treatment of neutrophils with anti-CD14 antibody under the presence of serum. Increase in the steady-state IL-8 mRNA level was concomitantly observed by stimulation of neutrophils with 500 ng/ml of P. gingivalis LPS under the absence of serum. These results indicate that P. gingivalis LPS activates NF-κB and AP-1 in both serum-dependent and -independent manners, followed by increased IL-8 transcription in neutrophils, and suggested a role for P. gingivalis LPS in IL-8 synthesis by neutrophils in inflamed gingiva and GCF.

Biased expression of T cell receptor Vβ genes in periodontitis patients

The aim of the present study was to investigate the hypothesis that there is selective activation and expansion of a limited repertoire of T cell receptor (TCR)‐bearing T cells in periodontitis tissue. We studied TCR Vβ gene expression in gingival tissues of periodontitis lesions and compared these with peripheral blood mononuclear cells (PBMC) from the same patients using polymerase chain reaction (PCR) amplification with 22 Vβ‐specific sense primers in combination with a common antisense Cβ‐specific primer. After initial therapy, gingival tissue specimens were obtained from 14 periodontitis patients at the time of periodontal surgery, and PBMC were also obtained from the same patients by density gradient centrifugation. Total RNA was extracted and analysed by reverse transcription PCR. The PCR products were electrophoresed on a 2% agarose gel and stained with ethidium bromide. For each product, gingival tissue and the respective peripheral blood were compared. The TCR repertoire identified in the PBMC in general overlapped with that of the gingival tissue. However, Vβ6 appeared to be over‐expressed in the gingival tissues compared with the PBMC, but Vβ16 appeared to be under‐expressed in the gingival tissues. Although it is not known whether this is due to antigen‐specific activation or superantigen activation, the data suggest that there may be as yet uncharacterized T cell subsets in periodontal disease tissue.

Immunoreactive Localization of Transforming Growth Factor-β Type II Receptor-Positive Cells in Rat Tibiae

To identify the target cells of transforming growth factor-beta (TGF-beta) in normal bone tissue, we examined TGF-beta type II receptor expression using immunohistochemistry, reverse transcription-polymerase chain reaction (RT-PCR), and in situ hybridization in young rat tibiae. In the epiphyseal growth plate, the TGF-beta type II receptor cDNA was detected by RT-PCR and, alternatively, the TGF-beta type II receptor protein and mRNA expression were observed in the chondrocytes in the lower part of the proliferative cell layer, and in maturative and hypertrophic cell layers by immunohistochemistry and in situ hybridization. Of these, proliferative and maturative chondrocytes, in particular, revealed strong mRNA expression. In the cortical bone area, immunoreactivity for the TGF-beta type II receptor was detected in the fibroblastic cells near the osteoblasts on the endosteal surface of cortical bone. In conclusion, our findings suggest that target cells of TGF-beta in normal bone tissue could be considered mainly as extracellular matrix-producing chondrocytes and undifferentiated preosteoblasts, and TGF-beta may affect matrix production and differentiation of these cells.