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Objective. Anti-endothelial cell antibodies (AECAs), which recognize a number of endothelial antigens, are seen in patients with systemic autoimmune diseases, more often in the presence of vasculitis than in its absence. Some AECAs induce apoptosis of endothelial cells (ECs), but their target antigens remain unknown. The aim of this study was to determine whether Hsp60 is a target antigen and whether AECAs induce apoptosis in ECs.Methods. Two-dimensional electrophoresis and conventional Western blotting techniques were used to characterize AECA targets. Hsp60 reactivity was determined by enzyme-linked immunosorbent assay.Results. Hsp60 was shown to be targeted by a proportion of AECAs. The level of reactivity was higher in patients with systemic autoimmune disease and vasculitis than in those without vasculitis and in patients with systemic lupus erythematosus than in patients with other systemic autoimmune diseases. Hsp60 was expressed on the plasma membrane of heat-stressed ECs, and this followed Hsp60 messenger RNA transcription, confinement of the protein to the cytoplasm, and translocation of the protein to the surface. Shedding of Hsp60 from ECs was induced by stress and resulted in the binding of soluble Hsp60 to the surface of ECs, particularly stressed ECs. Apoptosis of ECs was triggered by anti-Hsp60-containing AECA-positive sera and was inhibited by preincubation of the ECs with recombinant Hsp60.Conclusion. Our data support the notion that Hsp60 is an important target for AECAs and that such an interaction contributes to pathogenic effects, especially in vasculitis-associated systemic autoimmune disease.

As a result of damaging endothelial cells (ECs), Mycobacterium leprae triggers the production of antibodies (Abs). These anti-EC Abs (AECAs) can be divided into two types. The first type nonspecifically reacts with components of the cytosol (CY) and can be detected by enzyme-linked immunosorbent assay (ELISA). The second specifically reacts with the EC membrane (MB) and requires fluorescence-activated cell sorter (FACS) analysis to be detected. The presence of both types of AECAs was determined in 68 leprosy patients. The ELISA was positive for 35 of them but also for 30 of 34 malaria patients and 17 of 50 healthy African controls. However, whereas FACS analysis showed MB reactivity in only three malaria patients and four controls, this reactivity was found in 27 leprosy patients, more of those having the lepromatous than the tuberculoid form. Specificity for MB, which we failed to absorb by incubation with CY lysates, predominated over that for CY in leprosy, unlike malaria, where the EC reactivity was restricted to the CY. Western blot analysis and twodimensional electrophoresis revealed that calreticulin, vimentin, tubulin, and heat shock protein 70 were targeted by AECAs from leprosy patients, but other proteins remained unidentified. These auto-Abs, but not those from malaria patients, did activate ECs, as indicated by the E-selectin and intercellular adhesion molecule 1 upregulation, and/or induced them into apoptosis, as documented by four different methods. Our findings suggest that, in some but not all leprosy patients, AECAs may play a role in pathogenesis.

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