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Establishment of Porphyromonas gingivalis Heat-shock-protein-specific T-cell Lines from Atherosclerosis Patients

¹ Department of Periodontology, School of Dentistry,
² Department of Thoracic and Cardiovascular Surgery, School of Medicine,
³ Department of Molecular Biology, College of Natural Sciences, and
⁴ Department of Pharmacology, School of Medicine, Pusan National University, 1-10, Ami-Dong, Seo-Ku, Pusan 602-739, Korea;

*corresponding author, jilchoi{at}pusan.ac.kr

	ABSTRACT

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Human atherosclerotic plaques contain heat-shock proteins which may serve as potential targets of the immune response in atherosclerosis. Since periodontal infections are suggested as risk factors for the development of cardiovascular diseases, we undertook the present study to evaluate the T-cell immune responses specific to Porphyromonas gingivalis (P. gingivalis) heat-shock protein (hsp)60 in patients suffering from atherosclerosis. Anti-P. gingivalis hsp60 IgG antibody titers were elevated in all patients. We could establish P. gingivalis hsp-specific T-cell lines from the atheroma lesions and the peripheral blood. The T-cell lines were a mixture of CD4⁺ and CD8⁺ cells producing the cytokines characteristic of both Th1 and Th2 subsets. The present findings suggest that the T-cell immune response specific to P. gingivalis hsp60 may be involved in the immunopathologic process of atherosclerotic diseases.

KEY WORDS: Porphyromonas gingivalis • heat-shock protein • periodontal pathogen • atherosclerosis • T-cell line

	INTRODUCTION

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Periodontal infection may be one of the risk factors for cardiovascular diseases (Ando et al., 1995; Chiu, 1999; Haraszthy et al., 2000; Choi et al., 2001a; Okuda et al., 2001). This association is supported by the recent observation that Porphyromonas gingivalis can invade the endothelial cells (Deshpande et al., 1998). A strong causal relationship needs to be established (Slots, 1998; Slavkin, 1999), including the biological mechanisms by which periodontal disease could lead to coronary heart disease. Our recent report on P. gingivalis-specific T-cell lines in atheroma lesions supported a direct causal relationship between the two disease entities (Choi et al., 2001a). Mycobacterial heat-shock protein (hsp)65 or human hsp60 is thought to cause atherosclerosis (Wick et al., 1995; Mori et al., 2000), and T-cell immune responses specific to bacterial or human hsp have been demonstrated in atherosclerosis (Kaufmann et al., 1990; Ross, 1993; Wick et al., 1995). Based on these findings, it can be hypothesized that P. gingivalis can circulate in the blood and invade the arterial endothelium, where hsp's stimulate expression of pro-inflammatory cytokines and recruit T-lymphocytes, macrophages, and monocytes in atherosclerotic lesions. To elucidate how stress proteins contribute in the immunopathogenesis of atherosclerosis, we have attempted to establish T-cell lines specific for P. gingivalis hsp in human atheroma lesions and peripheral blood from atherosclerosis patients.

	MATERIALS & METHODS

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Systemic and Periodontal Examination of Atherosclerosis Patients
Patients with atherosclerosis were examined for systemic and periodontal diseases, including laboratory profiles. Informed consent forms were obtained for the surgical procedure according to the Guidelines of the Institutional Review Board of Pusan National University Hospital. Control groups included subjects without a history of atherosclerosis or periodontal disease and atherosclerosis patients without active periodontal disease.

Purification of Recombinant P. gingivalis hsp 60
GroEL gene cloned from P. gingivalis 381 (Maeda et al., 1994) was a gift from Professor Yoji Murayama, Okayama University Dental School, Japan. P. gingivalis GroEL gene was introduced into a glutathoine S-transferase-P. gingivalis GroEL fusion construct in the pGEX-4T-3 expression vector. The fusion construct was transformed into HB-1142 cells, and protein expression was induced by the addition of 1 M isopropyl â-D-thiogalactoside (IPTG). Cells were harvested and re-suspended in 100 mM triethanolamine-HCl, 170 mM NaCl, 1% Triton X-100, 10 mM dithiothreitol, pH 7.4, followed by sonication. A slurry of glutathione sepharose 4B beads (Pharmacia, Uppsala, Sweden), equilibrated twice with phosphate-buffered saline + 1% Triton X-100, was added to each tube and incubated. The beads were pelleted, re-suspended, and washed gently in phosphate-buffered saline + 1% Triton X-100. The fusion protein was eluted from the beads with elution buffer (10 mM reduced glutathione in 50 mM Tris-HCl, pH 8.0).

Measurement of Serum IgG Antibody Titers by ELISA
Microtiter plates were coated in triplicate with hsp (10 µg/mL) diluted in phosphate buffer (Choi et al., 2000, 2001a,b). The plates were washed, and an aliquot of serum samples serially diluted was added and incubated. The plates were washed, and peroxidase-conjugated mouse anti-human IgG (H+L) (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) was added. After 2 hrs of incubation, the plates were washed, and an aliquot of tetramethylbenzidine (Kirkegaard and Perry Laboratories, Gaithersburg, MD, USA) was added for incubation followed by the addition of 0.18 M H₂SO₄ to stop the reaction. Optical densities read at 450 nm of wavelength were plotted as a function of the serum dilution factor. One of the control group I sera (periodontally healthy subjects without history of cardiovascular diseases) was assigned an ELISA unit of 100 and serum IgG titer of the other control group I subjects, and the atherosclerosis patients were calculated. Antibody titer was considered to be elevated if it was higher than the control titer + 3x standard deviation.

Establishment and Characterization of P. gingivalis Heat-shock-protein-specific T-cell Lines
Atheroma lesions were collected and digested with collagenase (10 µg/mL, Boehringer-Mannheim, Berlin, Germany) so that a mononuclear cell population could be obtained (Choi et al., 2000, 2001a,b). We isolated peripheral blood mononuclear cells by a gradient cell separation technique using Ficoll-Paque medium (Pharmacia). Using 12-well tissue culture plates (Costar, Corning, NY, USA), we stimulated mononuclear cells from either atheroma lesions or peripheral blood with P. gingivalis together with antigen-presenting cells (APC) after treatment with mitomycin C. Peripheral blood lymphocytes were drawn from the patients, and non-T-cell fraction was used as APCs. After 2 wks of incubation, T-cells were allowed to rest for 1 wk. After the resting period, fresh mitomycin-treated non-T-cells and P. gingivalis were added again to induce T-cell proliferation.

From P. gingivalis-specific T-cell lines, P. gingivalis hsp-specific T-cell lines were established in the same manner by the addition of the heat-shock-protein antigen (5 µg/well in 12-well culture plates) in alternating cycles to stimulate T-cell lines. Culture supernatants were harvested and preserved at –20°C until used for cytokine assay. For characterization of T-cell lines, cells were double-stained with Per-CP-conjugated mouse anti-human CD3, FITC-conjugated mouse anti-human CD4, or PE-conjugated mouse anti-human CD8 monoclonal antibodies (PharMingen, San Diego, CA, USA). Phenotypic expression of each T-cell line was screened by flow cytometry by means of an Epics Elite ESP (Coulter, Hialeah, FL, USA).

T-cell Proliferation Assay
To evaluate the antigen-specific T-cell proliferation, we incubated P. gingivalis hsp-specific T-cells from each T-cell line established from the atheroma lesions with increasing amounts of P. gingivalis hsp antigen (0.1, 1, 10 µg/well) in triplicate (Choi et al., 2000). For antigen specificity testing, we added Mycobacterium tuberculosis hsp65 (Lionex GmbH, Braunschweig, Germany) in the same manner as a control antigen. Antigen-presenting cells treated with mitomycin C were added to each well. After 72 hrs of incubation, ³H-thymidine (1 µCi/well) was added, and cells were incubated for an additional 24 hrs for radioactivity assay.

Determination of Cytokine Concentrations
Briefly, 96-well plates (Costar, Corning, NY, USA) were coated with mouse anti-human IFN-, IL-4, or IL-10 (PharMingen; 4 µg/mL) diluted in a sodium carbonate buffer overnight at 4°C (Choi et al., 2000, 2001a,b). After being washed 3x with PBS/Tween, wells were blocked by PBS + 10% fetal bovine serum (PBS/FBS) and then washed 3x with PBS/Tween. Each sample and standard recombinant human IFN-, IL-4, or IL-10 (PharMingen), or PBS diluted in PBS/FBS + 0.05% Tween20 (PBS/FBS/Tween) were added, respectively, as the positive or negative control and incubated for 3 hrs. After the plates were washed, biotinylated mouse anti-human IFN-, IL-4, or IL-10 (PharMingen; 2 µg/mL in PBS/FBS/Tween) was added and incubated for 1 hr at room temperature. After plates were washed 4x with PBS/Tween, hydroperoxidase-conjugated streptavidin (PharMingen) were added and incubated. The plates were washed, and o-phenylenediamine (1 mg/mL in 0.1 M citrate buffer, pH 4.5) was added. A 4-N quantity of H₂SO₄ was added to stop the reaction. The optical densities were read at a wavelength of 490 nm. Optical densities of standard cytokines were plotted against the dilution factors, and the cytokine concentration of each sample was determined.

Statistical Analysis
To compare the IgG titer of the test group with those of control groups, we performed Student's t test.

	RESULTS

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Six patients diagnosed as having arteriosclerosis as well as severe periodontitis participated in the study. All were males, aged between 57 and 73 yrs, and smokers. There were nine atherosclerosis patients who did not demonstrate a history of destructive periodontal disease. They were aged between 53 and 75 yrs, and two-thirds of them were smokers. Detailed clinical profiles are presented in Table 1. Anti-P. gingivalis hsp60 IgG antibody titers of the six atherosclerosis patients with periodontitis were elevated, while those of nine atherosclerosis patients without periodontitis were not elevated when compared with those of ten healthy subjects (Table 2).

CD3⁺/CD4⁺ or CD3⁺/CD8⁺ T-cells of P. gingivalis hsp-specific T-cell lines from atheroma lesions demonstrated various phenotypic profiles between patients (ranging from 51.0 to 68.8% for CD4⁺ and from 17.4 to 40.1% for CD8⁺ T-cells), respectively (Fig., upper panel), whereas those from peripheral blood also demonstrated a range of phenotypic profiles between patients (ranging from 51.4 to 66.8% for CD4⁺ and from 16.2 to 35.6% for CD8⁺ T-cells), respectively (Fig., lower panel). P. gingivalis hsp-specific T-cell lines established from atheroma lesions showed in vitro proliferating activities in a dose-dependent manner when stimulated with 0.1, 1, and 10 µg/mL P. gingivalis hsp60 (Table 3), but not with M. tuberculosis hsp65 antigen.

View larger version (54K): [in this window] [in a new window]   Figure. Phenotype profiles of P. gingivalis hsp60-specific T-cell lines established from the atheroma lesion or peripheral blood from six patients analyzed by flow cytometry. The upper panel shows profiles of proportions (%) of CD3+/CD4+ T-cells (left) and CD3+/CD8+ T-cells (right) of T-cell lines established from atheroma lesions, while the lower panel indicates those of T-cell lines established from peripheral blood. The numbers 1-6 indicate each patient.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS & METHODS RESULTS DISCUSSION REFERENCES

Anti-P. gingivalis hsp60 IgG antibody titers in six patients in the experimental group were moderately elevated when compared with the healthy control subjects. However, we did not observe this phenomenon in nine atherosclerosis patients without periodontitis, who maintained clinically healthy periodontium. This observation indicates that a history of P. gingivalis infection in the subgingival area may contribute to the elevated humoral response to the bacterial hsp in the atherosclerosis patients.

We could successfully establish P. gingivalis- and P. gingivalis hsp-specific T-cell lines from mononuclear cells isolated from either the atheroma lesion or the peripheral blood of all six patients in the experimental group. However, we failed to establish T-cell lines responsive to other periodontopathic bacteria, including Actinobacillus actinomycetemcomitans, Capnocytophaga sputigena, and Eikenella corrodens. Moreover, none of the P. gingivalis hsp-specific T-cell lines proliferated in vitro with M. tuberculosis hsp65 antigen. T-cells from nine atherosclerosis patients without periodontitis, however, did not respond to P. gingivalis or any other bacteria tested. Percentile profiles of CD4⁺ or CD8⁺ T-cell lines from atheroma lesions or peripheral blood were very similar within the same patient. Taken together, these observations suggest that T-cells of the atherosclerosis patients who have experienced P. gingivalis infection might have been primed by circulating P. gingivalis hsp antigen and home to the atheroma lesion where P. gingivalis has infiltrated. To clarify this mechanism, we are investigating the repertoire for T-cell receptors.

P. gingivalis hsp-specific T-cell lines were a mixture of CD4⁺ and CD8⁺ cells producing the cytokines characteristic of both Th1 and Th2 subsets. To the best of our knowledge, this is the first report that P. gingivalis hsp-specific T-cell lines could be established in patients with atherosclerosis. In the present study, the specificity of T-cell proliferation to P. gingivalis hsp antigen was confirmed by four independent observations: that P. gingivalis hsp-specific T-cell lines were established from P. gingivalis-specific T-cell lines that did not respond to any other periodontopathic bacteria tested in the study; that all the P. gingivalis hsp-specific T-cell lines proliferated in a dose-dependent manner when stimulated in vitro with P. gingivalis hsp antigen; that none of the P. gingivalis hsp-specific T-cell lines proliferated in vitro with M. tuberculosis hsp65 antigen; and that T-cells from nine atherosclerosis patients without periodontitis did not respond to P. gingivalis or other bacteria.

	ACKNOWLEDGMENTS

 
The study was supported in part by a Grant #1999-2-20500-004-3 from KOSEF and by the 2002 research grant from Pusan National University Hospital.

Received May 14, 2001; Last revision February 25, 2002; Accepted February 25, 2002

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Choi JI, Borrello M, Smith E, Cutler CW, Sojar H, Zauderer M (2001b). Prior exposure of mice to Fusobacterium nucleatum modulates host response to Porphyromonas gingivalis.Oral Microbiol Immunol 16:338–344.[Medline]

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