The immune responses to human and microbial heat shock proteins in periodontal disease with and without coronary heart disease

Abstract

The human 60 kDa and microbial 65 kDa heat shock proteins (HSP) have been implicated in the pathogenesis of chronic periodontitis (P) and coronary heart disease (CHD). We have studied four male non-smoking cohorts of 81 subjects, matched for age. Group (a) consisted of a healthy group with minimal gingivitis (n = 18), group (b) were patients with P (n = 23), group (c) patients with CHD and minimal gingivitis (n = 20) and group (d) patients with CHD and P (n = 20). T cells separated from peripheral blood were found to be primed to both microbial HSP65 and human HSP60 but significant CD4, human leucocyte antigen (HLA) class II-restricted proliferative responses were found only with the human HSP60 in patients with P (P < 0.001) and CHD without (P < 0.001) or with (P < 0.00001) periodontitis. Dose-dependent inhibition of T cell proliferative responses was carried out to determine the receptors involved in recognition of HSP60 and HSP65. Monoclonal antibodies to CD14 showed inhibition of T cell proliferation stimulated by both HSP60 and HSP65, consistent with the role of CD14 as a receptor for these HSPs in P and CHD. The toll-like receptor 2 (TLR-) and TLR-4 were then studied and these showed that TLR-4 was recognized by microbial HSP65, whereas TLR-2 was recognised by human HSP60 in both P and CHD. However, a dissociation was found in the HSP60 and TLR4 interaction, as TLR4 appeared to have been recognized by HSP60 in P but not in CHD. The results suggest an autoimmune or cross-reactive CD4(+) class II-restricted T cell response to the human HSP60 in P and CHD. Further studies are required to determine if there is a common epitope within HSP60 that stimulates T cell proliferation in P and CHD.

Fig. 1

Lymphoproliferative responses of peripheral blood mononuclear cells (PBMC) stimulated by microbial heat shock protein (HSP)65 (a) and human HSP60 (b) in healthy controls, periodontitis, coronary heart disease (CHD)-G and CHD-P. *P < 0·01; **P < 0·001; ***P < 0·0001; ****P < 0·00001.

Fig. 2

T cell proliferative responses to microbial heat shock protein (HSP)65 (a), human HSP60 (b) before (open bars) and after treatment (hatched bars) with proteinase K or (c) BAPTA-AM and compared with lipopolysaccharide (LPS) before (grey bars) and after (hatched bars) treatment with proteinase K.

Fig. 3

Mean (± s.e.m.) stimulation indices of T cells (grey bars), enriched CD4 (open bars) and CD8 (cross-hatched bars) T cells stimulated with microbial heat shock protein (HSP)65 (a), human HSP60 (b), cells in healthy controls (n = 6), chronic periodontitis (P, n = 6) and coronary heart disease with gingivitis [coronary heart disease (CHD)-G, n = 6] or coronary heart disease with periodontitis (CHD-P, n = 6); *P < 0·001.

Fig. 4

Inhibition of T cell proliferation with monoclonal antibody (mAb) to human leucocyte antigen (HLA) class I, HLA class II or isotype controls of peripheral blood mononuclear cells (PBMC) stimulated with microbial heat shock protein (HSP)65 (a), human HSP60 (b) in five patients each with periodontitis (i), coronary heart disease (CHD)-G (ii) and CHD-P (iii).

Fig. 5

Inhibition T cell proliferation with monoclonal antibody (mAb) to CD14 of peripheral blood mononuclear cells (PBMC) stimulated with microbial heat shock protein (HSP)65 (a), human HSP60 (b), in patients each with periodontitis (i), coronary heart disease (CHD)-G (ii) and using monoclonal antibody (mAb) recognizing Toll-like receptor (TLR)2, TLR4 or isotype controls in periodontitis (iii) and CHD (iv); mean ± s.e.m. of seven patients in each group. mAb to HSP65 and HSP60 were also added to the cultures to confirm the antigen-specificity of this lymphoproliferation.