Induction of endothelial cell apoptosis by heat-shock protein 60-reactive antibodies from anti-endothelial cell autoantibody-positive systemic lupus erythematosus patients
Induction of endothelial cell apoptosis by heat-shock protein 60-reactive antibodies from anti-endothelial cell autoantibody-positive systemic lupus erythematosus patients
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DOI：
10.1002/art.20564
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发表时间：
2004-10-01
影响因子：
--
通讯作者：
Raymond, Y
中科院分区：
文献类型：
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作者：
Dieudé, M;Senécal, JL;Raymond, Y
Objective.确定抗磷脂综合征系统性红斑狼疮（SLE）患者的抗内皮细胞自身抗体（AECA）是否参与初始内皮细胞（EC）膜扰动效应，该效应被假定为提供抗磷脂抗体（aPL）结合的靶点，从而触发血栓级联反应。目的鉴定内皮细胞上的AECA抗原靶点，并确定EC膜被破坏的机制。通过流式细胞术测定SLE患者的AECA与EC的结合。通过免疫印迹法测定阳性AECA，并通过质谱法鉴定共有抗原。以重组形式测试该候选抗原的AECA识别。AECA在该抗原上亲和纯化，并与EC孵育以确定其生理效应。酶联免疫吸附试验测定抗Hsp60抗体滴度。分析抗Hsp60抗体和狼疮抗凝物（LAC）状态与发病至末次随访期间血栓形成的关系。73%的SLE患者血清具有与内皮细胞表面结合的IgG。这些阳性IgG对60 kd EC表面多肽具有反应性，该多肽被鉴定为人Hsp60。使用来自商业来源的抗Hsp60抗体或从结合EC的SLE血清亲和纯化的抗Hsp60抗体建立EC表面处Hsp60的存在。孵育的EC与这些抗Hsp60抗体诱导细胞凋亡的时间和剂量依赖性的方式，所确定的Hoechst 33342染料染色的浓缩核和膜联蛋白V结合表面磷脂酰丝氨酸。抗Hsp60抗体并不局限于SLE患者，也见于其他自身免疫性疾病患者。然而，当抗Hsp60抗体与LAC联合应用时，SLE患者血栓形成的发生率明显增加。Hsp60在内皮细胞表面的存在作为SLE血清中抗Hsp60抗体的靶标。这些抗Hsp60抗体与EC结合并诱导细胞凋亡，特别是磷脂酰丝氨酸暴露，从而为aPL的结合提供靶标并诱导随后的血栓级联反应。
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.