Iwanami Keiichi, Matsumoto Isao, Tanaka Yoko, Inoue Asuka, Goto Daisuke, Ito Satoshi, Tsutsumi Akito, Sumida Takayuki
Department of Clinical Immunology, Doctoral Program in Clinical Sciences, Graduate School of Comprehensive Human Science, University of Tsukuba, Tsukuba, Japan.
Arthritis Res Ther. 2008;10(6):R130. doi: 10.1186/ar2545. Epub 2008 Nov 7.
Arthritis induced by immunisation with glucose-6-phosphate isomerase (GPI) in DBA/1 mice was proven to be T helper (Th) 17 dependent. We undertook this study to identify GPI-specific T cell epitopes in DBA/1 mice (H-2q) and investigate the mechanisms of arthritis generation.
For epitope mapping, the binding motif of the major histocompatibility complex (MHC) class II (I-Aq) from DBA/1 mice was identified from the amino acid sequence of T cell epitopes and candidate peptides of T cell epitopes in GPI-induced arthritis were synthesised. Human GPI-primed CD4+ T cells and antigen-presenting cells (APCs) were co-cultured with each synthetic peptide and the cytokine production was measured by ELISA to identify the major epitopes. Synthetic peptides were immunised in DBA/1 mice to investigate whether arthritis could be induced by peptides. After immunisation with the major epitope, anti-interleukin (IL) 17 monoclonal antibody (mAb) was injected to monitor arthritis score. To investigate the mechanisms of arthritis induced by a major epitope, cross-reactivity to mouse GPI peptide was analysed by flow cytometry and anti-GPI antibodies were measured by ELISA. Deposition of anti-GPI antibodies on the cartilage surface was detected by immunohistology.
We selected 32 types of peptides as core sequences from the human GPI 558 amino acid sequence, which binds the binding motif, and synthesised 25 kinds of 20-mer peptides for screening, each containing the core sequence at its centre. By epitope mapping, human GPI325-339 was found to induce interferon (IFN) gamma and IL-17 production most prominently. Immunisation with human GPI325-339 could induce polyarthritis similar to arthritis induced by human GPI protein, and administration of anti-IL-17 mAb significantly ameliorated arthritis (p < 0.01). Th17 cells primed with human GPI325-339 cross-reacted with mouse GPI325-339, and led B cells to produce anti-mouse GPI antibodies, which were deposited on cartilage surface.
Human GPI325-339 was identified as a major epitope in GPI-induced arthritis, and proved to have the potential to induce polyarthritis. Understanding the pathological mechanism of arthritis induced by an immune reaction to a single short peptide could help elucidate the pathogenic mechanisms of autoimmune arthritis.
已证实DBA/1小鼠中由葡萄糖-6-磷酸异构酶（GPI）免疫诱导的关节炎依赖于辅助性T细胞（Th）17。我们开展这项研究以鉴定DBA/1小鼠（H-2q）中GPI特异性T细胞表位，并研究关节炎产生的机制。
对于表位定位，从T细胞表位的氨基酸序列中鉴定出DBA/1小鼠主要组织相容性复合体（MHC）II类（I-Aq）的结合基序，并合成了GPI诱导性关节炎中T细胞表位的候选肽。将人GPI致敏的CD4+ T细胞和抗原呈递细胞（APC）与每种合成肽共培养，并通过酶联免疫吸附测定（ELISA）测量细胞因子产生情况以鉴定主要表位。将合成肽免疫DBA/1小鼠以研究肽是否可诱导关节炎。用主要表位免疫后，注射抗白细胞介素（IL）-17单克隆抗体（mAb）以监测关节炎评分。为研究主要表位诱导关节炎的机制，通过流式细胞术分析与小鼠GPI肽的交叉反应性，并通过ELISA测量抗GPI抗体。通过免疫组织学检测抗GPI抗体在软骨表面的沉积。
我们从人GPI 558个氨基酸序列中选择了32种肽作为核心序列，其与结合基序结合，并合成了用于筛选的25种20聚体肽，每种肽在其中心包含核心序列。通过表位定位，发现人GPI325-339最显著地诱导干扰素（IFN）γ和IL-17产生。用人GPI325-339免疫可诱导出与人GPI蛋白诱导的关节炎相似的多关节炎，并且给予抗IL-17 mAb可显著改善关节炎（p < 0.01）。用人GPI325-339致敏的Th17细胞与小鼠GPI325-339发生交叉反应，并导致B细胞产生抗小鼠GPI抗体，这些抗体沉积在软骨表面。
人GPI325-339被鉴定为GPI诱导性关节炎中的主要表位，并被证明具有诱导多关节炎的潜力。了解对单个短肽的免疫反应诱导关节炎的病理机制可能有助于阐明自身免疫性关节炎的发病机制。
