THE MECHANISM OF NEPHRITOGENIC ANTI-DNA ANTIBODY PRODUCTION IN LUPUS NEPHRITIS

本研究的目的是阐明人致肾炎性抗DNA抗体产生的抗原触发因素。我们发现，人单克隆致肾炎O-81抗DNA抗体结合到内质网应激诱导蛋白，Herp.1。分别用DNA固定化琼脂糖和Herp固定化琼脂糖从活动性狼疮肾炎患者中获得抗DNA和抗Herp抗体。狼疮抗DNA抗体与疱疹病毒结合。此外，抗Herp抗体显示与DNA的特异性结合。每只6周龄的BALB/c雌性小鼠经腹腔注射Herp或Hen Egg Lysozyme（HEL）进行免疫。HEL免疫BALB/c小鼠后，可诱导抗dsDNA和抗ssDNA抗体产生，并在肾小球内形成IgG沉积. SLE患者和健康人的外周血淋巴细胞用抗Herp单克隆抗体染色。活动期SLE患者外周血淋巴细胞自发表达Herp，而健康人外周血淋巴细胞自发表达Herp。在无任何刺激的条件下，体外培养72小时，观察SLE PBLs中Herp的表达。在健康PBL中没有观察到Herp的表达。为了评估Herp的抗原性，将PBL与Herp一起培养6天，并通过ELISPOT测定来测量产生抗DNA抗体的克隆的数目。在体外用Herp刺激后，活动期SLE患者的PBL中产生抗DNA抗体的克隆数增加，但用HEL或dsDNA刺激后没有增加。SLE患者和健康人的PBL对Herp刺激无反应，提示细胞内可诱导的自身蛋白可能是诱发人肾源性抗DNA自身抗体的触发因子，其中可能存在ER应激。

The aim of the present study is to elucidate the antigenic trigger for the production of human nephritogenic anti-DNA antibodies. We found that a human monoclonal nephritogenic O-81 anti-DNA antibody bound to an endoplasmic reticulum stress-inducible protein, Herp.1. The anti-DNA or anti-Herp antibodies were obtained from the patients with active lupus nephritis by using a DNA-immobilized or a Herp-immobilized agarose, respectively. Lupus anti-DNA antibodies bound to Herp. Moreover, the anti-Herp antibodies showed specific binding to DNA.2. Each 6-week-old BALB/c female mouse was immunized intraperitoneally with Herp or Hen Egg Lysozyme (HEL). immunization with Herp, but not HEL, elicited anti-dsDNA as well as anti-ssDNA antibodies in BALB/c mice and formed the deposits of IgG in the renal glomeruli.3. The PBLs from SLE patients or healthy subjects were stained with a monoclonal anti-Herp antibody. Herp was spontaneously expressed in the PBLs of patients with active SLE, but in the PBLs of healthy subjects. The expression of Herp in the SLE PBLs was observed during the 72 hours in vitro culture without any stimulation. No expression of Herp was observed in healthy PBLs.4. To assess the antigenicity of Herp, the PBLs were cultured with Herp for 6 days, and the number of anti-DNA antibody producing clones was measured by ELISPOT assay. The number of anti-DNA antibody producing clones in the PBLs from active SLE patients increased in vitro after stimulation with Herp, but not with HEL or dsDNA. The PBLs form inactive SLE patients or healthy subjects did not respond to the stimulation with Herp.These results imply that an inducible intracellular self protein could be a trigger for human nephritogenic anti-DNA autoantibodies, where ER stress may be noted.