Surface staining and cytotoxic activity of heat-shock protein 60 antibody in stressed aortic endothelial cells.

Abstract

Heat-shock protein (hsp) expression can be induced by high temperature, exposure to cytokines or oxygen radicals, ischemia, hemodynamic overload, or viral infections. To determine whether surface expression of hsp60 occurs in aortic endothelial cells stressed by high temperature or cytokines, cells from rat aortas were cultivated and stained with several types of monoclonal antibodies against hsp60. Other antibodies, eg, those against intercellular adhesion molecule-1 (ICAM-1), or immune response-associated antigens were also used as controls. Positive staining of endothelial cells on the surface and in the cytoplasm was observed after pretreatment of the cells with cytokine-containing medium, tumor necrosis factor-alpha (TNF-alpha), or interleukin-1 alpha and labeling with a specific monoclonal antibody against hsp60 (II-13). Fluorescence-activated cell sorter analyses showed that over 80% of living endothelial cells stressed by cytokine-containing medium, by TNF-alpha, or at 42 degrees C, but not by interleukin-1 alpha, were positively surface stained with this antibody. Increased intensity of immunostaining with antibodies to ICAM-1 and immune response-associated antigen was also seen on the cytokine-stressed endothelial cells. Furthermore, when TNF-alpha stimulated endothelial cells labeled with 51Cr were incubated with antibody II-13 in the presence of complement, significant lysis occurred. In summary, endothelial cells stressed by high temperature or certain cytokines, eg, TNF-alpha, express hsp60 in the cytoplasm and on their surfaces, and these cells were susceptible to complement-dependent lysis by hsp60-specific antibody. These observations may be significant for elucidating the mechanisms of the involvement of immune reactions to hsp65/60 in initiating atherosclerosis.

Association of autoantibodies to heat‐shock protein 60 with arterial vascular events in patients with antiphospholipid antibodies

“…Heat-shock proteins show considerable sequence homology among species, and immune reactions to Hsp60 in infections by microorganisms that express Hsp65 have been widely described (3). Anti-Hsp65 antibodies, induced in response to these pathogens, can cross-react with self Hsp60 expressed on endothelial cells (4)(5)(6)(7). Elevated levels of anti-Hsp60 have been associated with progression and severity of atherosclerosis (8)(9)(10)(11)(12)(13)(14)(15), with vasculitis in systemic autoimmune diseases (16), and with thrombotic events in the context of systemic lupus erythematosus (SLE) and lupus anticoagulant (LAC) positivity (17).…”

Cardiovascular disease

“…31,32 However, it is possible that heat shock proteins potentiate later stages of atherogenesis by inducing heat shock protein antibodies, 5,6,22 by the recruitment of heat shock proteinspecific inflammatory lymphocyte populations, 20 or by their expression on the endothelial cell surface. 10 The putative involvement of heat shock proteins in the establishment and progression of the atherosclerotic lesion is undoubtedly complex. Heat shock protein expression may be secondary to the inflammatory process, in that the expression of cytokines by both the vascular endothelium and infiltrating leukocyte populations drives the expression or release of heat shock proteins from the vessel wall.…”

“…7 Although heat shock proteins are typically regarded as being intracellular, they can be expressed on the surface of mononuclear cells 8,9 and stressed aortic endothelial cells. 10 They can also be released from cultured rat glial and human islet cells, 11,12 and Hsp60 and Hsp70 have been identified in the serum of healthy individuals. 13,14 Given the potential involvement of heat shock proteins in the pathogenesis of vascular disease and the association between hypertension and atherosclerosis, 15 we determined the levels of circulating Hsp60; Hsp70; and anti-human Hsp60, anti-human Hsp70, and anti-mycobacterial 65-kDa protein antibodies in normotensive (NT) and BHT subjects.…”

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

“…It is known, that HSP60, or at least parts of it, may be exposed on the cell surface [4][5][6]. Consequently, the next steps are to confirm the presence of this epitope within early atherosclerotic lesions and whether immune reactions against this epitope are involved in pathogenesis of atherosclerosis.…”

“…Since microbial HSP60s serve as major antigens in protection from, and pathogenesis of infectious diseases, autoimmune disorders such as rheumatoid arthritis, systemic sclerosis, psoriasis, Kawasaki's disease or Behcet's disease, are thought to be triggered by shared B-and T-cell epitopes cross-reactive between eukaryotic and prokaryotic HSP60 [3]. Interestingly, HSP60, which is physiologically active inside mitochondria, was found also on the surface of stressed eukaryotic cells, where it may serve as a danger signal and as target-(auto)-antigen for immune reaction [4][5][6].…”