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

Adam Hasan, Danesi Sadoh, Bret Jones

Queen Mary University of London, London, UK.
Unit of Periodontology, King’s College London Dental Institute, London, UK.

DOI: 10.4236/jibtva.2014.31001                

Abstract

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.

Keywords

Heat Shock Proteins; Periodontitis; Cardiovascular Disease; Cytokines; IL-17

1. Introduction

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 [1]. There is evidence from cross-sectional studies implicating a multitude of organisms [2,3]. HSPs are highly conserved proteins [4,5], expressed in all eukaryotic and prokaryotic cells and involved in both innate and adaptive immune responses, with innate responses helping to focus the cellular immune responses [6]. The high degree of homology between microbial and human HSPs has led to the hypothesis that tissue damage can occur as a result of cross-reactivity between bacterial and human HSPs. Humoral and T cell responses to HSP60/65 have been demonstrated in CP [4,5,7-9].

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 [10]. TH1 cells produce IL-2 and IFN-γ and facilitate cell mediated immune responses and are pro-inflammatory. In contrast, TH2 cells produce IL-4, IL-5 and IL-10 [11] simultaneously inhibiting TH1 responses and promoting humoral immunity. The histological picture of stable periodontitis lesions resembles a type IV delayed hypersensitivity response and in progressive lesions changes to lesions dominated by B cells and plasma cells [12]. These findings have been previously interpreted as meaning the stable lesion is mediated by TH1 cells and the progressive lesion by TH2 cells [12]. Animal studies suggest that Th1 are involved in the progression of chronic periodontitis [13-16]. However, several studies using human cells have reported a predominance of TH2 responses in patients with CP compared to control patients [17-19]. These conflicting results may be due in part to differences in materials examined and methodology used.

There is an association between CP and atherosclerosis (cardiovascular disease) and an increased risk of developing cardiovascular disease in patients who have CP [20]. TH1 cytokines are found to be predominantly involved in the pathogenesis of atherosclerosis [21,22]. The TH1 subpopulation of CD4 + T cells predominates among atheroma plaque infiltrating lymphocytes [21,22] and becomes activated to secrete IFN-γ, IL-2, TNF-α, TNF-β [21-23], and IL-18 [23,24]. Upregulation of IFN- γ, IL-2, TNF-α, and TNF-β results in the activation of macrophages and vascular cells, promoting inflammation and cellular immunity [25]. IL-18 and its receptor act in concert to induce the production of IFN-γ, by smooth muscle cells [23]. Most human atherosclerotic lesions produce little TH2 type cytokines such as IL-4, IL-5 and IL-10 [21,22], except for IL-6 [26]. Furthermore, IL-6 correlates with CRP and plaque size in patients with acute myocardial infarction [27].

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 [32]. In addition, human HSP60 has been shown to induce PBMC to produce IL-4, IL-10 [34] and IFN-γ [19,34] in patients with rheumatoid arthritis and chronic periodontitis [35].

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 [36]. We have previously found autoimmune or cross-reactive CD4+ class II-restricted T cell responses to the human HSP60 in CP and CHD [8]. Given the potential for innate immunity to focus adaptive immune responses, the objective of this study was to investigate the role of HSP-mediated induction of intracellular TNF- α, IFN-γ, and IL-2, IL-4 and IL-5, and IL-17 from PBMC of patients with chronic periodontitis and coronary heart disease, and to determine if these responses are related to inflammation, as reflected by CRP levels.

2. Material and Methods

2.1. Subjects

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 (Table 1). All subjects were aged between 38 and 58 years of age and had at least 15 standing teeth. Subjects were excluded from the study if they had systemic disease including diabetes mellitus, recurrent aphthous stomatitis, autoimmune disease, a history of malignancies, previous treatment for periodontitis, or a history of antibiotic therapy within the past six months prior to recruitment. Subjects were allocated into 4 groups: gingivitis, CP, Ging/CHD or CP/CHD. In each subject, probing depths and recession of all teeth were determined using a William’s probe. Recession was measured as the distance from the cemento-enamel junction to the gingival margin. Measurements were taken from 6 sites; probing attachment loss was then calculated as the sum of probing pocket depth and recession. Periodontitis was defined as probing attachment loss ³4 mm in at least 4 teeth and CG group as probing attachment loss <2 mm in all teeth. Ethical committee approval was obtained (code no 98/12/04) and subject consent obtained. 40 ml of venous blood were withdrawn from each subject.

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.

2.2. HSP

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.

Table 1. Demographic data of patients showing median, interquartile range, mean and standard error of mean (SEM) of age, smoking status, smoking pack years and probing attachment level.

^#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.

2.3. Separation of PBMC

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.

2.4. Detection of Intracellular Cytokines by Flow Cytometry

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 (Figure 1) in a humidified atmosphere (5% CO₂ at 37˚C). To investigate the role of LPS contamination some of the patients PBMC were further incubated with heat treated LPS or HSP by boiling 100˚C for 30 minutes. After 18 hours of incubation, cells were transferred into 5 ml round-bottom tubes (Falcon 352,058). Cells were stained using CD4 fluorescein isothiocyanate (FITC) 5 µl (BD Pharmingen, San Diego), CD14 and CD8 peridinin chlorophyll-protein (PerCP) 5 µl (BD Pharmingen, San Diego), then incubated in the dark at room temperature for 30 minutes. Cells were then centrifuged at 500 g for 5 minutes and washed once in 2 mls of PBS/azide (×1 PBS, 0.5% BSA, 0.1% sodium azide). Cells were fixed with 1 ml FACS lysing solution containing <50% Diethylene glycol and < 15% formaldehyde (BD Pharmingen 349,202) diluted 1:10 with PBS/azide and incubated at room temperature for 10 minutes. The cells were washed in PBS/azide cells and then permeabilised with 500 µl of FACS permeabilising solution (BD pharmingen 340,973) containing 0.02% saponin, diluted 1:10 with deionised water. Cells were incubated for 10 minutes at room temperature and then centrifuged at 500 g for 5 minutes, washed once and resuspended in PBS/azide.

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-

(a)(b)(c)(d)(e)(f)(g)

Figure 1. (a)-(g): Histogram of flow cytometric intracellular cytokine expression in monocytes stained with PE-labelled anticytokine monoclonal in a patient with chronic periodontitis antibody showing events versus mean fluorescence intensity. (a) TNF-µ expression following stimulation with LPS; (b) TNF-µ expression following stimulation with microbial HSP65; (c) TNF-µ expression following stimulation with Human HSP60; (d) TNF-µ expression following heat treatment with LPS; (e) TNF-µ expression following stimulation with heat treated microbial HSP65; (f) TNF-µ expression following stimulation with heat treated human HSP60; (g) TNF-µ expression following incubation with medium only. (a) LPS TNF-µ; (b) HSP65 TNF-µ; (c) HSP60 TNF-µ; (d) LPS TNF-a following heat treatment; (e) Heat denatured microbial HSP65 TNF-µ; (f) Heat denatured human HSP60 TNF-µ; (g) Medium TNF-µ.

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).

2.5. Statistical Method

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 (r_s); 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.

2.6. Detection of C-Reactive Protein by ELISA

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.

3. Results

3.1. Detection of Intracellular Cytokines

Unstimulated cells incubated in medium showed relatively low mean fluorescence intensity (MFLI) (Figure 2(g)) in all intracellular cytokines studied; no statistically significant difference was found amongst the 4 groups (ANOVA, p = 0.803, Table 2).

There was a statistically significant difference amongst the 4 groups in MFLI for intracellular TNF-α by ANOVA (p = 0.001) (Table 2, Figures 2 and 3); also between the gingivitis group compared to the CP group (p = 0.017) and between the gingivitis group and the ging/CHD

Figure 2. Detection of intracellular TNF-a in monocytes stimulated with human HSP60 showing median, interquartile range, mean and standard error of mean (SEM) of MFLI for each of 4 groups of patients (ANOVA p = 0.001) o maximum; × 75^th percentile;  minimum;   25^th percentile;median.

Conflicts of Interest

The authors declare no conflicts of interest.

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