Induction of endothelial cell apoptosis by heat‐shock protein 60–reactive antibodies from anti–endothelial cell autoantibody–positive systemic lupus erythematosus patients

Abstract

Objective. To determine whether anti-endothelial cell autoantibodies (AECAs) from systemic lupus erythematosus (SLE) patients with the antiphospholipid syndrome are involved in the initial endothelial cell (EC) membrane perturbation effect that is postulated to provide a target for antiphospholipid antibody (aPL) binding and, hence, to trigger the thrombotic cascade. To identify the AECA antigenic target on ECs and to determine the mechanism whereby the EC membrane is disrupted.Methods. AECAs from SLE patients were assayed for binding to ECs by flow cytometry. Positive AECAs were assayed by immunoblotting, and a consensus antigen was identified by mass spectrometry. This candidate antigen was tested in recombinant form for AECA recognition. AECAs were affinity-purified on this antigen and incubated with ECs to determine their physiologic effects. Anti-Hsp60 antibody titers were determined by enzyme-linked immunosorbent assay. The relationship of anti-Hsp60 status and lupus anticoagulant (LAC) status to thrombotic manifestations between disease onset and the last followup visit were analyzed.Results. Most of the SLE sera (73%) possessed IgG that bound to the surface of ECs. These positive IgG shared reactivity against a 60-kd EC surface polypeptide that was identified as human Hsp60. The presence of Hsp60 at the EC surface was established using anti-Hsp60 antibodies from commercial sources or affinity-purified from SLE sera that bound ECs. Incubation of ECs with these anti-Hsp60 antibodies induced apoptosis in a time-and dose-dependent manner, as determined by Hoechst 33342 dye staining of condensed nuclei and by annexin V binding to surface phosphatidylserine. Anti-Hsp60 antibodies were not restricted to SLE patients, but were found in patients with other autoimmune diseases. However, anti-Hsp60 antibodies were significantly associated with an increased frequency of thrombosis when present in combination with LAC in the SLE patients.Conclusion. The presence of Hsp60 at the surface of ECs serves as a target for the anti-Hsp60 antibodies in SLE sera. These anti-Hsp60 antibodies bind to ECs and induce apoptosis, particularly phosphatidylserine exposure, thus providing a target for the binding of aPL and inducing the subsequent thrombotic cascade.

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

“…Anti-Hsp60 titers were also determined in 41 control sera, and the value corresponding to the 75th percentile in the control group (OD 450 0.834) was used to define the cutoff for elevated IgG anti-Hsp60. This cutoff value has been shown previously to distinguish between low and high titers of antiHsp60 in healthy controls, patients with SLE, and patients with other rheumatic diseases (17). Figure 1 shows anti-Hsp60 levels in 41 healthy controls and in the 402 participants in the Figure 1.…”

“…Differential endothelial cell expression of Hsp60 may at least partially explain the greater susceptibility of the arterial bed to antiHsp60. We and others have provided both in vitro and in vivo evidence that the presence of anti-Hsp60 is itself sufficient to cause endothelial cell dysfunction (apoptosis, activation, and/or injury) (2,(16)(17)(18). Such antiHsp60-induced endothelial damage would constitute the "first hit" and would favor the recruitment of aPL by exposing phosphatidylserine, a negatively charged phospholipid that interacts with phospholipid-binding proteins.…”

“…Anti-Hsp60 titers were determined by enzyme-linked immunosorbent assay as described previously (17), with the following modifications: 1) the antigen was recombinant human Hsp60 (StressMarq Biosciences), 2) antibodies were detected using horseradish peroxidase-conjugated goat anti-human IgG (Southern Biotechnology), and 3) plates were developed with the BD OptEIA TMB Substrate Reagent set (BD Biosciences) and quantitated by reading the optical density at 450 nm (OD 450 ) (EL800 reader; BioTek Instruments). Anti-Hsp60 titers were also determined in 41 control sera, and the value corresponding to the 75th percentile in the control group (OD 450 0.834) was used to define the cutoff for elevated IgG anti-Hsp60.…”

“…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). Moreover, anti-Hsp60 from SLE patients were shown to bind to the surface of endothelial cells and induce apoptosis in these cells (17).…”

Association Between Panel Reactive Antibody and Antiendothelial Cell Antibody Positivity in Kidney Transplant Patients

“…10,11 The AECAs may trigger an inflammatory process and induce endothelial cell apoptosis and necrosis. 12 The AECAs are detected in a wide range of clinical pathologies that involve the vascular system, including autoimmune disease, infectious disease, and vasculopathies, but they also can be observed in healthy individuals. 13,14 Alloimmune-mediated injury of the endothelium leads to the aberrant expression of self proteins and subsequent generation of autoantibodies.…”