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RESEARCH REPORTS |
1 Department of Periodontology and Research Institute for Oral Biotechology, School of Dentistry, 2 Department of Thoracic and Cardiovascular Surgery, School of Medicine, 3 Department of Molecular Biology, College of Natural Sciences, 4 Department of Pharmacology, and 5 Department of Microbiology, School of Medicine, Pusan National University, 1–10, Ami-Dong, Seo-Ku, Pusan 602–739, Korea;
* corresponding author, jrapa{at}pusan.ac.kr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: Porphyromonas gingivalis • heat-shock protein • epitope • atherosclerosis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Due to a considerably high degree of sequence homology between bacterial and human heat-shock proteins (HSP), this protein might be involved in autoimmune disease mechanisms operating in humans (Hansson, 2001; Wick et al., 2001). 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). The host immune system primed by HSP of a major periodontal pathogen, such as Porphyromonas gingivalis (P. gingivalis), can cross-react with its cognate mammalian counterpart in gingival connective tissue or arterial walls (Yamazaki et al., 2002). To provide evidence that P. gingivalis may be actively involved in the immunopathogenic process of atherosclerosis, we have recently reported T-cell responses specific to P. gingivalis or P. gingivalis HSP in atherosclerosis patients (Choi et al., 2001a, 2002).
In clarifying the immune mechanisms modulating the autoimmune diseases, it is critical that one identify the immunodominant epitope(s) of an infecting pathogen that is (are) exclusively recognized by T- and/or B-cells. In the present study, we have attempted to characterize P. gingivalis HSP-reactive T-cell lines, and to identify T- and/or B-cell epitopes of P. gingivalis HSP and human HSP.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Measurement of Anti-P. gingivalis, Anti-P. gingivalis HSP60, and Anti-human HSP60 Serum IgG Antibody Titers
Recombinant P. gingivalis HSP60 was produced and purified from the P. gingivalis GroEL gene (Maeda et al., 1994), and the purity was verified as previously described (Choi et al., 2002). Microtiter plates coated with either formalinized P. gingivalis cells, P. gingivalis HSP60, or human HSP60 (StressGen, Victoria, BC, Canada), diluted in 10 mM phosphate buffer (Choi et al., 2000, 2001a, Choi et al., b), were incubated with an aliquot of serum samples. After samples were washed, peroxidase-conjugated mouse anti-human IgG (H+L) (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) was added. 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 were plotted as a function of the serum dilution factor for determination of the titer. Antibody titer was considered to be elevated if it was higher than the mean control titer + 3x the standard deviation.
Western Immunoblot
Recombinant P. gingivalis HSP60 or human HSP60 was subject to SDS-PAGE and electro-transferred to nitrocellulose membrane. After the membrane was blocked, human sera were added for incubation. The membrane was washed, followed by the addition of horseradish-peroxidase-conjugated mouse anti-human IgG (H+L) (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). After the membrane was washed, tetramethylbenzidine was added for color development. For identification of cross-reactivity of bacterial HSP among periodontopathogenic bacteria, bacterial cell lysates of heat-shocked P. gingivalis, Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Prevotella intermedia, Treponema denticola, and Fusobacterium nucleatum were subject to immunoblot with mouse anti-P. gingivalis HSP antisera.
Synthetic Peptide
We synthesized a total of 108 decapeptides, spanning the entire amino acid sequence of P. gingivalis GroEL and human HSP60, respectively, using an Epitope-Scanning Kit (Chiron Mimotopes, Clayton, Victoria, Australia). Peptides were designed to overlap by 5 amino acid residues.
T-cell Epitope Mapping
T-cells (1 x 105 cells/well) from previously established P. gingivalis HSP-reactive T-cell lines (Choi et al., 2002) were inclubated with synthetic peptides (5 µg/mL) of P. gingivalis HSP60 and antigen-presenting cells (5 x 106 cells/well). After 48 hrs of incubation, the cells were labeled with 3H-thymidine for an additional 6 hrs of incubation and counted in a liquid scintillation counter. Proliferation was presented as the stimulation index (SI): the ratio of the mean counts per minute (cpm) with antigen to the cpm without antigen. SI values of 3 or greater were considered to be positive.
Preparation of Conjugated Plate
Conjugation of the synthetic peptide of P. gingivalis HSP60 or human HSP60 to the microtiter plate (CovaLink plate, NUNC, Roskilde, Denmark) was done with a water-soluble 1-ethyl-(3-dimethyl-aminopropyl) (EDC) carbodiimide in the presence of N-hydroxy-succinamide (NHS). Each peptide was dissolved in dimethyl sulfoxide, diluted with 0.1 M carbonate-bicarbonate buffer, and used for coating plates.
B-cell Epitope Mapping
The conjugated plate was washed, and aliquots of serum samples were added and incubated. After the plates were washed, peroxidase-conjugated mouse anti-human IgG (H+L) (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) was added. The plates were incubated and 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. Optical density means and the standard deviations of the ELISA signals to the peptides were calculated for each sample. The mean ± 3 standard deviation was assigned as the baseline, and each signal to the peptide was assigned a positive or negative response.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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).
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). Sera from healthy controls reacted neither with P. gingivalis HSP60 nor with human HSP60. In addition, mouse anti-P. gingivalis HSP60 antisera recognized P. gingivalis HSP and cross-reacted with all the HSPs induced by heat-shock treatment of putative periodontopathogenic bacteria tested (Fig. 1B).
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. Fifteen antigenic peptides that showed positive signals in more than four out of six patients were designated as major B-cell epitopes of P. gingivalis HSP60 (peptides no. 3, 6, 15, 29, 33, 39, 53, 56, 69, 74, 75, 85, 93, 99, and 102). Of these, 5 were identical to T-cell epitopes (Table 2
). In the same manner, major B-cell epitopes of human HSP60 were identified, being peptides no. 15, 29, 53, 56, 69, and 74 (Table 2).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Mean anti-P. gingivalis, anti-P. gingivalis HSP60, or human HSP60 IgG antibody titers in six atherosclerosis patients were higher when compared with the control subjects. Sera from atherosclerosis patients who had elevated IgG antibody to P. gingivalis HSP60 demonstrated cross-reactivity with human HSP60, as evidenced by Western blot results, suggesting that the autoimmune pathogenic mechanisms were regulated by P. gingivalis HSP in human atherosclerosis (Hansson, 2001; Wick et al., 2001; Choi et al., 2002; Yamazaki et al., 2002). Moreover, mouse anti-P. gingivalis HSP60 antisera recognized and reacted with P. gingivalis HSP as well as with all other HSPs induced by heat treatment of putative periodontopathogenic bacteria tested. Several authors have claimed the critical role of bacterial stress proteins or human HSP in recruiting immune cells which target antigens, consequently leading to the development of plaque and atheroma lesions (Kaufmann et al., 1990; Ross, 1993; Wick et al., 1995).
We performed the present study to scan anti-HSP60 serum antibody for specific linear B-cell epitopes on the P. gingivalis HSP60 proteins and compared them with T-cell epitopes. Interestingly, 5 of these epitopes were identified as common T-cell and B-cell epitopes in atherosclerosis patients (Table 2). When B-cell epitopes for corresponding sequences of P. gingivalis HSP60 or human HSP60 were compared, peptides no. 15, 29, 53, 56, 69, and 74 of P. gingivalis HSP60 and human HSP60 were identified as cross-reactive B-cell epitopes. Of these, peptide no. 15 of P. gingivalis HSP60 (VKEVASKTND) has also been identified as a B-cell epitope in periodontitis (Maeda et al., 2000), and the corresponding peptide no. 15 of human HSP60 (VQDVANNTNE) has been identified as a B-cell epitope in atherosclerosis (Metzler et al., 1997) and a T-cell epitope in atherosclerosis (unpublished observation). Of additional interest, peptide no. 56 of P. gingivalis HSP60 (PGFGDRRKAM) and human HSP60 (PGFGDNRKNQ), respectively, was consistently identified as a cross-reactive B-cell epitope in all the patients. This peptide has also been identified as a B-cell epitope in periodontitis (Maeda et al., 2000). These immunodominant cross-reactive epitopes might be critical in the immunopathogenic process in human atherosclerosis and could thus be candidate peptides for prospective vaccine development against atherosclerosis. We have established the epitope-specific T-cell lines from human atheromatous plaque to transfer adoptively into severe combined immunodeficiency (SCID) mice. This would eventually allow one to evaluate human immune responses for vaccine efficacy testing in experimental atherogenesis.
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ACKNOWLEDGMENTS |
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Received May 10, 2003; Last revision August 28, 2004; Accepted September 14, 2004
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REFERENCES |
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