Background-Work from our laboratory has proven that increased titers of anti-heat shock protein 60 (HSP60) antibodies are associated with atherosclerosis and that HSP60-reactive T-cells are present in atherosclerotic lesions. Recent studies from others demonstrated that HSP60 directly activates endothelial cells and macrophages. Methods and Results-To explore the possibility that HSP60 exists in the circulation, where it could exert its functions, we performed a population-based study with 826 subjects aged 40 to 79 years. The following items were measured in all participants: serum soluble HSP60 (sHSP60); anti-Escherichia coli lipopolysaccharide; anti-HSP65, anti-Chlamydia, and anti-Helicobacter pylori antibodies; and a variety of acute phase reactants (C-reactive protein, ␣ 1 -antitrypsin, and ceruloplasmin) and markers of systemic inflammation. Carotid atherosclerosis was assessed twice (1990 and 1995), and 15 other risk factors were evaluated. Our data show that levels of sHSP60 were significantly elevated in subjects with prevalent/incident carotid atherosclerosis and that these levels were correlated with common carotid artery intima/media thickness. Multiple logistic regression analysis documented these associations as independent of age, sex, and other risk factors. Interestingly, sHSP60 was also correlated with anti-lipopolysaccharide, anti-Chlamydia and anti-HSP60 antibodies, various markers of inflammation, and the presence of chronic infections. The risk of atherosclerosis associated with high sHSP60 levels was amplified when subjects had clinical and/or laboratory evidence of chronic infections. Conclusions-Our data provide the first evidence of a strong correlation between sHSP60 and atherosclerosis, suggesting that sHSP60 may play important roles in activating vascular cells and the immune system during the development of atherosclerosis. (Circulation. 2000;102:14-20.)
The highly conserved and ubiquitous heat shock proteins (HSP) are essential for the cellular homeostasis and efficiently trigger cellular responses to stress conditions. Both microbial and human HSP act as dominant antigens in numerous infectious and autoimmune diseases such as atherosclerosis, inducing a strong immune-inflammatory response. In the present study, the surface localization of HSP60 on stressed and unstressed human umbilical venous endothelial cells (HUVECs) was investigated using sensitive high resolution microscopy methods and flow cytometry. Confocal laser scanning microscopy (CLSM) revealed an increase of HSP60 in the mitochondria and on the surface of heat-stressed living and fixed HUVECs compared to unstressed cells. Atomic force microscopy (AFM), which has developed as sensitive surface-probe technique in biology, confirmed the presence of HSP60 on the membrane of stressed cells at an even higher lateral resolution by detecting specific single molecule binding events between the monoclonal antibody AbII-13 tethered to AFM tips and HSP60 molecules on cells. The interaction force (force required to break a single AbII-13/HSP60 bond) was 59±2 pN, which correlated nicely to the 51±1 pN measured with isolated HSP60 attached to mica surfaces. Overall, we found clear evidence for the occurrence of HSP60 on the surface of stressed HUVECs in a very similar patchy distribution pattern in living and fixed cells. The relevance of our findings with respect to the role of HSP60 in atherogenesis is discussed.
Objective-Growing evidence suggests that immune reactions to heat shock protein 60 (HSP60) are involved in atherogenesis. Because of the high phylogenetic conservation between microbial and human HSP60, bacterial infections might be responsible for breaking the tolerance to self-HSP60, which is expressed on the surface of stressed arterial endothelial cells. Methods and Results-We purified serum antibodies to Escherichia coli HSP60 (GroEL), the 60-kD chlamydial HSP, and HSP65 of Mycobacterium tuberculosis by affinity chromatography from clinically healthy subjects with sonographically proven carotid atherosclerosis. Reactivity of the purified antibodies with overlapping human HSP60 peptides was measured, and 8 shared common epitopes, recognized by all anti-bacterial HSP60/65 antibodies, were identified. Antisera specific for these cross-reactive epitopes were produced by immunizing rabbits with peptides derived from human HSP60. By immunohistochemistry, the epitopes were found to be present in the arterial wall of young subjects during the earliest stages of the disease. Conclusions-Antibodies to microbial HSP60/65 recognize specific epitopes on human HSP60. These cross-reactive epitopes were shown to serve as autoimmune targets in incipient atherosclerosis and might provide further insights into the mechanisms of early atherogenesis. Key Words: epitopes Ⅲ autoimmunity Ⅲ atherosclerosis Ⅲ heat shock protein 60 Ⅲ aging T he current theory of atherosclerosis suggests that the first stages can be described as an inflammatory process in the arterial wall. 1 However, the initial triggering event and the involved (auto)antigens still remain controversial. Heat shock proteins of the 60-kDa family (HSP60) are among 1 of the most highly conserved families of proteins. 2 Prokaryotic HSP60s have homology Ͼ97% at their protein levels, whereas prokaryotic and human HSP60 (hHSP60) have Ͼ70% amino acid sequence homology. Although microbial HSP60s serve as major antigens in protection from and pathogenesis of infectious diseases, several autoimmune disorders, like rheumatoid arthritis, systemic sclerosis, psoriasis, Kawasaki disease, and Behcet's disease, are believed to be triggered by shared B-and T-cell epitopes that are crossreactive between eukaryotic and prokaryotic HSP60. 3 HSP60 can be found not only in mitochondria but also on the surface of stressed eukaryotic cells. 4 -6 Because chronic bacterial infections are known to be a risk factor for atherosclerosis, 7,8 immunologic cross-reactions between bacterial HSP60 and hHSP60, which is expressed on the surface of stressed arterial cells, might be involved in atherogenesis. In a prospective, longitudinal study, we demonstrated that elevated levels of anti-mycobacterial HSP65 (mHSP65) antibodies, crossreacting with hHSP60, served as a prognostic marker for the incidence, prevalence, severity, and progression of carotid atherosclerosis in a clinically healthy population. 9,10 Those results have subsequently been confirmed by others. 11,12 Recent evidence corroborates that...
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