Cytokines Elicited by HSP60 in Periodontitis with and without Coronary Heart Disease

The human 60 kDa and microbial 65 kDa heat shock proteins (HSP) have been implicated in the pathogenesis of chronic periodontitis (CP) and coronary heart disease (CHD). We have studied 100 subjects: Group (a) consisted of patients with gingivitis (n = 25), group (b) were patients with CP (n = 25), group (c) patients with CHD and gingivitis (n = 25) and group (d) patients with CHD and CP (n = 25). PBMCs separated from peripheral blood were stimulated with medium, PMA/ionomycin, human HSP60, microbial HSP65, or no stimulus for 18 hours before intracellular IL-2, IFN-γ, TNF-α, IL-4, IL-5, or IL-17 were detected by flow cytometry. The mean fluorescence intensity (MFLI) for intracellular TNF-α was significantly increased when PBMC were stimulated with human HSP60 amongst the four groups (p = 0.001, ANOVA);pairwise comparisons revealed significant differences in MFLI between the gingivitis group and the CP (p = 0.017);between gingivitis and ging/CHD (p = 0.001) as well;but no significant difference between the CP and CP/CHD (p = 0.442). There was no significant difference in intracellular expression of IL-17, or any of the other cytokines tested;and the MFLI for HSP-stimulated were comparable to unstimulated cultures. When heat-labile human HSP60 was heated, intracellular cellular TNF-α expression was abrogated. In contrast, heat-stable LPS elicited TNF-α expression from monocytes in bulk cultures in all groups. These results suggest that the cytokine expression was dependent on human HSP60 and not LPS. Serum CRP was significantly associated with MFLI of intracellular TNF-α in CP patients (rs = 0.665, p = 0.026) and CP/CHD (rs = 0.699, p = 0.011). We conclude that human HSP60 elicits increased monocytic expression of TNF-α in patients with CP, CP/CHD or ging/CHD compared to patients with gingivitis. Since the marker of inflammation, namely CRP correlates with CP with or without CHD and not with mild chronic gingivitis or ging/CHD, this suggests that human HSP60-induced production of TNF-α is associated with CP and not CHD. There was no significant difference in intracellular expression of IL-17.

Immune Responses to HSP65/60 in Periodontal Disease

Chronic periodontitis (CP) is a chronic inflammatory condition which destroys the supporting tissues of teeth and increases in prevalence with age. Immune responses against heat shock proteins (HSP) can be cross-reactive among bacterial and human antigens. There is evidence that microbial HSP65 and human HSP60 are involved in periodontal disease. The aim of this study is to investigate immune responses to the human HSP60 and microbial HSP65 in patients with CP and relate these to the level of inflammation and smoking status. We collected serum samples from 30 patients with chronic gingivitis (CG) and 30 patients with CP. In each group, eight subjects were current smokers. ELISA was used to determine the levels of serum anti-HSP and C-reactive protein (CRP) in each group. Peripheral blood mononuclear cells were also isolated and stimulated with HSPs. Significant lymphoproliferation was seen in CP when stimulated with human HSP60. CRP and serum anti-human HSP60 IgG were elevated in CP compared to the CG, but not serum anti-microbial HSP 65 IgG. In view of the potential confounding effects of smoking in CP, a group of current smokers (n = 16) was also recruited to investigate whether smoking affects HSP immune responses. There was no significant difference in HSP-induced lymphoproliferation between smokers and non-smokers in either the CG or CP. There was a significant correlation between CRP and lymphoproliferative responses to Human HSP60 irrespective of smoking status. This study shows that serum anti-human HSP60 IgG and serum CRP are raised in untreated CP. In CP, serum CRP levels correlated significantly with human HSP60-induced lymphoproliferation, but not with anti-HSP antibody levels.