Surface staining and cytotoxic activity of heat-shock protein 60 antibody in stressed aortic endothelial cells.
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.
Xu
Q
Q
Institute for Biomedical Aging Research, Austrian Academy of Sciences, Innsbruck.
Schett
G
G
Seitz
C S
CS
Hu
Y
Y
Gupta
R S
RS
Wick
G
G
D000818:Animals; D000906:Antibodies / Q000032:analysis*; D000920:Antibody-Dependent Cell Cytotoxicity; D002478:Cells, Cultured; D018834:Chaperonin 60 / Q000276:immunology*; D016207:Cytokines / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000166:cytology* / Q000276:immunology; D005260:Female; D005455:Fluorescent Antibody Technique; D006358:Hot Temperature; D051381:Rats; D013194:Staining and Labeling; D013312:Stress, Physiological; D013499:Surface Properties
D000906:Antibodies; D018834:Chaperonin 60; D016207:Cytokines