Jethwa H S, Clarke S H, Itoh-Lindstrom Y, Falk R J, Jennette J C, Nachman P H
Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill 27599, USA.
J Immunol. 2000 Oct 1;165(7):3890-7. doi: 10.4049/jimmunol.165.7.3890.
Anti-neutrophil cytoplasmic Abs, directed primarily toward myeloperoxidase (MPO) and proteinase 3, are detected in the majority of patients with distinct forms of small vessel vasculitides and pauci-immune necrotizing glomerulonephritis. However, the origin of these autoantibodies remains unknown. We studied the V region gene use in murine anti-MPO Abs derived from Spontaneous Crescentic Glomerulonephritis/Kinjoh mice. A total of 13 anti-MPO-producing hybridomas were generated from four unimmunized mice. Ten of the 13 hybridomas (corresponding to 3 of 4 clones) expressed Vkappa1C but differed in their use of VH genes. The remaining three hybridomas expressed a Vkappa5 gene. Anti-MPO hybridomas from individual mice were derived from single clones as deduced by sequence similarity and splice-site identity. We found a statistically significant bias of amino acid replacement mutations to the complementarity-determining regions (CDR) in the Vkappa1C-expressing hybridomas. Intriguingly, all 10 Vkappa1C hybridomas share a lysine to glutamate mutation in the CDR1. To determine the effects of somatic V gene mutations on binding to MPO, we generated an anti-MPO Ab with an unmutated Vkappa1C L chain and compared its ability to bind MPO with its mutated counterpart. The mutated hybridoma-derived Ab has a 4.75-fold higher avidity for MPO than the unmutated Ab. These results suggest that: 1) the L chain plays a dominant role in determining Ab specificity to MPO, 2) the anti-MPO Ab response is oligoclonal, consistent with Ag selection, and 3) MPO is a driving Ag in the murine anti-MPO Ab response.
抗中性粒细胞胞浆抗体主要针对髓过氧化物酶（MPO）和蛋白酶3，在大多数患有不同形式的小血管炎和寡免疫坏死性肾小球肾炎的患者中均可检测到。然而，这些自身抗体的来源仍然未知。我们研究了源自自发性新月体性肾小球肾炎/金代小鼠的鼠抗MPO抗体中的V区基因使用情况。从四只未免疫的小鼠中总共产生了13个产生抗MPO的杂交瘤。13个杂交瘤中的10个（对应于4个克隆中的3个）表达Vκ1C，但在VH基因的使用上有所不同。其余三个杂交瘤表达Vκ5基因。通过序列相似性和剪接位点一致性推断，来自单个小鼠的抗MPO杂交瘤源自单个克隆。我们发现在表达Vκ1C的杂交瘤中，氨基酸替换突变在互补决定区（CDR）存在统计学上的显著偏向。有趣的是，所有10个Vκ1C杂交瘤在CDR1中都有赖氨酸到谷氨酸的突变。为了确定体细胞V基因突变对与MPO结合的影响，我们产生了一种具有未突变Vκ1C轻链的抗MPO抗体，并将其与MPO结合的能力与其突变对应物进行比较。突变的杂交瘤衍生抗体对MPO的亲和力比未突变抗体高4.75倍。这些结果表明：1）轻链在决定抗体对MPO的特异性方面起主导作用；2）抗MPO抗体反应是寡克隆的，与抗原选择一致；3）MPO是鼠抗MPO抗体反应中的驱动抗原。
