Autoantibodies against heat shock protein 60 mediate endothelial cytotoxicity.

Abstract

Stress or heat shock proteins (hsp) are a family of approximately two dozen proteins with a high degree of amino acid sequence homology between different species, ranging from prokaryotes to humans, and are representative of a generalized response to environmental and metabolic stressors. Our previous studies showed increased expression of human hsp6O on endothelial cells of arterial intima with atherosclerotic lesions, and elevated levels of serum antibodies (Ab) against hsp65/60 in subjects with carotid atherosclerosis. To investigate the possible involvement of anti-hsp65/60 Ab in endothelial injury, specific hsp-Ab were isolated from human high titer sera by affinity chromatography and probed on heat-shocked human umbilical vein endothelial cells. Purified human anti-hsp65/60 Ab reacted specifically with mycobacterial hsp65, human hsp60, and a 60-kD protein band of heat-shocked endothelial cells. (5, 6). As a matter of fact, immunization of rabbits with recombinant mycobacterial hsp65 induced the development of atherosclerotic lesions at characteristic predilection sites known to be subject to increased hemodynamic stress, such as the aortic and the branching of large vessels (7). A previous epidemiological study (8) from our laboratory had demonstrated that serum antibodies to mycobacterial hsp65, which is a homologue of human hsp6O, were significantly increased in clinically healthy subjects with sonographically demonstrable carotid atherosclerosis compared to those without lesions. This increased antibody level was independent of other established risk factors, such as hyperlipidemia, smoking, hypertension, diabetes mellitus, and obesity, thus providing evidence for a strong correlation of hsp65 antibodies with carotid atherosclerosis. These serum antibodies cross-reacted with recombinant human hsp6O homologue, and also with homogenates of atherosclerotic lesions both showing a 60-kD protein band on Western blots (9). Double-immunofluorescence labeling of atherosclerotic lesions with human anti-hsp65 sera and cell marker-specific antibodies demonstrated expression of hsp60 by endothelial cells, as well as by macrophages and smooth muscle cells therein. These findings indicated the existence of serum antibodies reacting not only with mycobacterial hsp65 but also with mammalian hsp60, expressed as an autologous cellular component (6,9).Based on these observations we next addressed the question whether human anti-hsp65/60 antibody (Ab) from high titer sera could react with surface proteins, e.g., hsp60 on stressed endothelial cells, and whether these antibodies could mediate 1. Abbreviations used in this paper: ADCC, antibody-dependent cellular cytotoxicity; hsp, heat shock protein.Schett, Xu, Amberger, Van der Zee, Recheis, Willeit, and Wick 2569 J. Clin. Invest.

Th1 polarized response induced by intramuscular DNA-HSP65 immunization is preserved in experimental atherosclerosis

“…Also in human studies, higher anti-Hsp65 titers have been associated with progression and severity of lesions (15,16). In addition, these antibodies, that show cross-reactivity with human Hsp60 and bacterial Hsp65, mediate cytotoxicity against endothelial cells (28)(29)(30).…”

Identification of atherosclerosis-associated conformational heat shock protein 60 epitopes by phage display and structural alignment

“…Purified human anti-bacterial HSP60 antibodies showed cytotoxicity on stressed, but not unstressed, human endothelial cells [19,32]. We recently reported linear (continuous) B-cell epitopes shared between microbial and human HSP60 [16].…”

“…For purification of anti-HSP antibodies, sera of five subjects with titers ≥1:1280 and sonographically proven atherosclerotic lesions in the carotid artery were pooled. Affinity chromatography of serum was performed using a previously described method [19]. Briefly, complement of pooled sera was heatinactivated and immunoglobulins were collected by standard ammonium sulphate precipitation.…”

Carta ao editor

“…Diversos trabalhos apontam os níveis elevados de anticorpos IgG contra hsp humana de 60kDa e micobacteriana de 65kDa com aumento da idade e doença arteriosclerótica na carótida (3)(4)(5)(6)(7)(8)11) , diferente do que menciona a Carta ao Editor, recém-enviada, em seu segundo parágrafo. Outrossim, as publicações pertinentes a esse tópico vêm crescendo na literatura médica.…”