STAINING OF ENDOTHELIAL-CELLS AND MACROPHAGES IN ATHEROSCLEROTIC LESIONS WITH HUMAN HEAT-SHOCK PROTEIN-REACTIVE ANTISERA

Our previous epidemiological studies have shown that levels of serum a ntibodies against mycobacterial heat-shock protein (hsp) 65 correlate positively with carotid atherosclerosis in subjects aged 40 to 79 year s. To determine whether these high-titer sera also react with homologo us human hsp60 and/or cell components of atherosclerotic lesions, we s elected 15 human sera samples, each with high or low titers to recombi nant mycobacterial hsp65, and investigated their reactivity with human arterial lesion components by immunoblotting and immunofluorescence t echniques. All five higher-titer sera against hsp65 reacted with a 60- kDa band of atherosclerotic lesion proteins and human recombinant hsp6 0 on Western blots. Pooled sera with low antibody titers to hsp65 dilu ted similarly as high-titer sera did not show reactivity with atherosc lerotic lesion and media proteins. By immunohistochemistry and immunof luorescence with human immunoglobulin G isolated from different sera, labeled with biotin, and visualized with a streptavidin conjugate, pos itive staining was observed in sections of fatty streaks and atheroscl erotic plaques of carotid arteries, and weak staining was observed in the normal intima. Double immunofluorescence identified the majority o f positively stained cells as macrophages, endothelial cells, and a fe w smooth muscle cells. In summary, serum antibodies against hsp65 cros s-react with the human 60-kDa homologue present in high levels in athe rosclerotic lesions and are mainly reacting with macrophages and endot helial cells, supporting our concept of a possible involvement of humo ral-mediated immune reaction against hsp60 in atherogenesis.