Ryan J J, Reynolds J, Norgan V A, Pusey C D
Renal Section, Division of Medicine, Imperial College School of Medicine, Hammersmith Hospital, London, UK.
Nephrol Dial Transplant. 2001 Feb;16(2):253-61. doi: 10.1093/ndt/16.2.253.
The autoantigen in Goodpasture's disease is known to be the non-collagenous domain of the alpha3 chain of type IV collagen, alpha 3(IV)NC1. There is mounting evidence that alpha 3(IV)NC1 is also a target of autoimmunity in experimental autoimmune glomerulonephritis (EAG). Sado et al. [Kidney Int 1998; 53, 664-671] have reported that recombinant human alpha 3(IV)NC1 and alpha4(IV)NC1 are nephritogenic in WKY rats. We have proposed that immunization with homologous antigen is more appropriate for detailed investigation of autoimmunity in EAG.
To this end, we have cloned and sequenced rat alpha 3(IV)NC1 and expressed it in COS-7 cells. Recombinant rat alpha 3(IV)NC1, secreted into the COS-7 cell supernatant, was purified on an anti-M2 FLAG affinity column and characterized by western blotting. Recombinant antigen was then used to immunize WKY rats, in order to induce EAG.
The recombinant material was antigenic as judged by binding to sera from patients with Goodpasture's disease and a mAb to alpha 3(IV)NC1. Immunization of WKY rats (n=5), with recombinant rat alpha 3(IV)NC1 in FCA at a dose of 1 mg/kg resulted in circulating anti-GBM antibodies directed towards alpha 3(IV)NC1, linear deposits of IgG on the GBM, albuminuria, deposits of fibrin in the glomeruli, severe focal necrotizing glomerulonephritis with crescent formation, and glomerular influx of CD8+ T cells and macrophages. Western blot analysis demonstrated that sera from these rats bound strongly to recombinant rat alpha 3(IV)NC1, as well as to collagenase-solubilized human and rat GBM. The pattern of binding was indistinguishable from that of sera from patients with Goodpasture's disease.
This purified recombinant rat alpha 3(IV)NC1, which is both antigenic and nephritogenic, will be of value in analysing autoimmune responses in experimental anti-GBM disease.
已知肺出血肾炎综合征（Goodpasture's disease）中的自身抗原是IV型胶原α3链的非胶原结构域，即α3(IV)NC1。越来越多的证据表明，α3(IV)NC1也是实验性自身免疫性肾小球肾炎（EAG）中的自身免疫靶点。Sado等人[《肾脏病国际》1998年；53卷，664-671页]报道，重组人α3(IV)NC1和α4(IV)NC1在WKY大鼠中具有致肾炎性。我们提出，用同源抗原进行免疫更适合对EAG中的自身免疫进行详细研究。
为此，我们克隆并测序了大鼠α3(IV)NC1，并在COS-7细胞中进行表达。分泌到COS-7细胞上清液中的重组大鼠α3(IV)NC1在抗M2 FLAG亲和柱上进行纯化，并通过蛋白质印迹法进行鉴定。然后用重组抗原免疫WKY大鼠，以诱导EAG。
通过与肺出血肾炎综合征患者血清及抗α3(IV)NC1单克隆抗体结合判断，重组物质具有抗原性。用剂量为1 mg/kg的重组大鼠α3(IV)NC1与弗氏完全佐剂（FCA）免疫WKY大鼠（n = 5），导致产生针对α3(IV)NC1的循环抗肾小球基底膜（GBM）抗体、IgG在GBM上的线性沉积、蛋白尿、纤维蛋白在肾小球中的沉积、伴有新月体形成的严重局灶坏死性肾小球肾炎以及CD8 + T细胞和巨噬细胞向肾小球内浸润。蛋白质印迹分析表明，这些大鼠的血清与重组大鼠α3(IV)NC1以及胶原酶溶解的人和大鼠GBM均有强烈结合。结合模式与肺出血肾炎综合征患者血清的结合模式无法区分。
这种纯化的重组大鼠α3(IV)NC1既具有抗原性又具有致肾炎性，在分析实验性抗GBM疾病中的自身免疫反应方面将具有重要价值。
