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.
Chronic periodontitis (CP) is an inflammatory disease characterised by connective tissue destruction and bone resorption, affecting 10% - 15% of the developed world population and is the major cause of tooth loss in adults [
In CP, there is controversy as to whether cells with a TH1 or TH2 cytokine profile are associated with disease progression, as both TH1 and TH2 cytokines are produced [
There is an association between CP and atherosclerosis (cardiovascular disease) and an increased risk of developing cardiovascular disease in patients who have CP [
Various studies have demonstrated that HSP60/65 can activate monocytes/macrophages to produce pro-inflammatory cytokines such as TNF-α [28-31], IL-6 [32,33] and IL-8 [
Cytokines produced by T-cells cannot always be easily categorised as either TH1 or TH2. TH17 cells, which produce IL-17 and have been implicated in autoimmune and chronic inflammatory conditions such as rheumatoid arthritis, may offer an alternative to the TH1/TH2 paradigm, however, their role in atherosclerosis is similarly unclear [
Patients with CG and CP were recruited from the Unit of Periodontology at Guy’s and St Thomas’ Foundation Trust and the Coronary Care Unit at St Thomas’ Hospital (
The gingivitis subjects had minimal gingival inflammation and bleeding on probing (percentage of bleeding sites, mean ± s.d., 1.8 ± 0.8), compared with the diseased groups, with bleeding sites in CP (48.9 ± 11.7), coronary heart disease with gingivitis (Ging/CHD; 1.9 ± 0.7) and CP/CHD (45.9 ± 8.7), All Ging/CHD patients had CHD as determined by greater than 60% diameter stenosis in at least two major epicardial coronary arteries, and conversely only eight of the healthy control subjects were confirmed to be free of CHD based on angiography, the remaining members of the group denying any chest pain symptoms.
Recombinant HSP65 derived from Mycobacterium bovis was prepared at the National Institute of Public Health and Environmental Protection, Bilthoven, the Netherlands and used at a predetermined optimal concentration of 10 µg/ml. Human HSP60 was purchased from Stressgen (Victoria, Canada). The two HSPs were detoxified using Detoxi-gel columns (Pierce, Oxford, UK) and the endotoxin level was determined by Limulus Amoebocyte Lysate assay (Sigma-Aldrich, Poole, Dorset, UK). The concentration of endotoxin was < 0.007 U/µg or 7 pg endotoxin/µg for both HSPs.
#significant p value for between group analysis of variance by Kruskal-Wallis test. *p value for pairwise comparison by Bonferroni between gingivitis and chronic periodontitis group. **p value for pairwise comparison by Bonferroni between gingivitis and gingivitis/CHD group. ***p value for pairwise comparison by Bonferroni between chronic periodontitis and chronic periodontis/CHD group. NS = never smokers ES = ex-smokers PS = present smokers IQ range = interquartile range.
Peripheral blood mononuclear cells (PBMC) were seperated from defibrinated blood samples by density gradient centrifugation (Nycomed Pharma As, Oslo, Norway). Blood was diluted 1:1 with tissue culture medium (RPMI 1640, Sigma-aldrich, UK) containing penicillin 100 mg/ml, streptomycin 100 U/ml (Gibco, Paisley, Scotland, UK), 2 mM L-glutamine (Sigma-aldrich, Irvine UK) at room temperature. This mixture was then layered onto an equal volume of lymphoprep (Ficoll-Paque TM PLUS, Amersham Biosciences, Uppsala Sweden) in 50 ml plastic tubes (Bibby Sterilin Staffordshire UK) and centrifuged at 600 g for 30 minutes at 20˚C. Mononuclear cells were removed from the interface, and washed twice by spinning at 200 g for 10 minutes. The serum was retained and diluted with medium containing penicillin 100 mg/ml, streptomycin 100 U/ml (Gibco, Paisley, Scotland, UK), 2 mM L-glutamine (Sigma-aldrich, UK) to 10% and used in cell cultures.
PBMC from 100 patients were cultured (5 × 105 cells/ well) in 24 well flat bottomed plates (Corning 25,820). Cytokine secretion by PBMC was blocked by adding monensin 4 mg/ml (Sigma-aldrich, Irvine UK) and brefeldin A 10 mg/ml (Sigma-aldrich, Irvine UK). PBMC were then activated with phorbol myristate acetate (PMA) 10 ng/ml plus ionomycin 1 µg/ml (Sigma-aldrich, Irvine UK), human HSP60 10 µg/ml (NSP-540 Stressgen UK), microbial HSP 65 10 µg/ml (NSP581 stressgen UK) or medium alone and incubated at previously predetermined optimal time of 18 hours (
Cells were then stained for intracellular cytokines, using 1 µg/ml of the following monoclonal antibodies conjugated to phycoerythrin (PE): anti-human IL-2 phycoerythrin (PE) conjugate (554,566 BD Pharmingen, San Diego), anti-human IL-4PE conjugate (554,516 BD Pharmingen, San Diego), anti-human IL-6PE conjugate (554,545 BD Pharmingen, San Diego), anti-human IFN-γ PE conjugate (554,701 BD Pharmingen, San Diego), anti-humanTNF-α PE conjugate (554,513 BD Pharmin-
gen, San Diego), anti-human IL-17PE conjugate (560,438 BD Pharmingen) or CD64PE conjugate (Serotec Oxford). After 30 minutes cells were washed twice and resuspended in PBS/Azide. Samples were analysed in a Coulter Epics FACS machine, at least 10,000 events were analysed. Results were expressed as the mean fluorescence intensity (MFLI).
Data recorded on dental charts and data obtained from the different experiments were transcribed onto computer records and analysed using SPSS-11.0 for windows (2001). All continuous variables were examined to establish whether the data conformed to a normal distribution with the Lilliefors (Kolmogorov-Smirnov) test for normality. Skewness and kurtosis were also examined. Analysis revealed non-normal distribution for all variables tested which were not amenable to log or square root transformation. Therefore the non-parametric KruskalWallis ANOVA test was used to assess differences between the groups established. The significance level was set at p < 0.05. Post ANOVA pairwise comparisons between the gingivitis group, CP group, ging/CHD group and CP with CP/CHD group were carried out with MannWhitney U test, using the Bonferroni correction. Interrelations between two variables were tested using the Spearman ranked correlation coefficients (rs); when the p value was <0.05, correction for smoking pack years was carried out. Non-parametric data are displayed in tables as median (1st and 3rd quartile). Data were also displayed as mean ± standard error of mean (SEM) to facilitate comparison with previously reported findings in the literature.
Pre-diluted standard (50 mL) and blank were added to a 96-well plate pre-coated with anti-serum CRP IgG (Kalon Biological Ltd). Serum samples were diluted 1:1000 with assay diluent (Kalon Biological Ltd.) and dispensed in duplicates to designated wells in CRP precoated plates. The plate was then incubated at room temperature for 60 minutes. Plates were washed 4 times with wash buffer (Kalon Biological Ltd); 100 mL of CRP tracer (affinity purified sheep anti-CRP labelled with alkaline phosphatase, Kalon Biological Ltd UK) were then dispensed to each well and incubated uncovered for 30 minutes at room temperature. Plates were washed again 4 times with washing buffer (Kalon Biological Ltd, UK).
Substrate solution (100 ml of 4-nitrophenylphosphate in substrate buffer Kalon Biological Ltd.) was then dispensed to each well and incubated at room temperature for 30 minutes. The reaction was stopped with 100 ml of (120 g/L) sodium hydroxide. Optical densities were read at 405 nm with microplate reader (Anthos 2001, Anthos labtec instruments UK). A standard curve was constructed with standard points and curve fitted with four parameter logistic curve fitting software. Test serum values were then read off the standard curve.
Unstimulated cells incubated in medium showed relatively low mean fluorescence intensity (MFLI) (
There was a statistically significant difference amongst the 4 groups in MFLI for intracellular TNF-α by ANOVA (p = 0.001) (