Division of Rheumatology, Department of Internal Medicine, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan.
Molecular Regulation of Aging, Tokyo Metropolitan Institute of Gerontology, Itabashi-ku, Japan.
Clin Exp Immunol. 2021 Mar;203(3):385-399. doi: 10.1111/cei.13556. Epub 2020 Dec 20.
The citrullinated inter-alpha-trypsin inhibitor heavy chain 4 (cit-ITIH4) was identified as its blood level was associated with the arthritis score in peptide glucose-6-phosphate-isomerase-induced arthritis (pGIA) mice and the disease activity in patients with rheumatoid arthritis (RA). This study aimed to clarify its citrullination pathway and function as related to neutrophils. In pGIA-afflicted joints, ITIH4 and cit-ITIH4 levels were examined by immunohistochemistry (IHC), immunoprecipitation (IP) and Western blotting (WB), while peptidylarginine deiminase (PAD) expression was measured by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), IHC and immunofluorescent methods. The pGIA mice received anti-lymphocyte antigen 6 complex locus G6D (Ly6G) antibodies to deplete neutrophils and the expression of cit-ITIH4 was investigated by WB. The amounts of ITIH4 and cit-ITIH4 in synovial fluid (SF) from RA and osteoarthritis (OA) patients were examined by I.P. and W.B. Recombinant ITIH4 and cit-ITIH4 were incubated with sera from healthy volunteers before its chemotactic ability and C5a level were evaluated using Boyden's chamber assay and enzyme-linked immunosorbent assay (ELISA). During peak arthritic phase, ITIH4 and cit-ITIH4 were increased in joints while PAD4 was over-expressed, especially in the infiltrating neutrophils of pGIA mice. Levels of cit-ITIH4 in plasma and joints significantly decreased upon neutrophil depletion. ITIH4 was specifically citrullinated in SF from RA patients compared with OA patients. Native ITIH4 inhibited neutrophilic migration and decreased C5a levels, while cit-ITIH4 increased its migration and C5a levels significantly. Cit-ITIH4 is generated mainly in inflamed joints by neutrophils via PAD4. Citrullination of ITIH4 may change its function to up-regulate neutrophilic migration by activating the complement cascade, exacerbating arthritis.
瓜氨酸化的细胞间α-胰蛋白酶抑制剂重链 4（cit-ITIH4）被鉴定为其血液水平与肽葡萄糖-6-磷酸异构酶诱导关节炎（pGIA）小鼠的关节炎评分以及类风湿关节炎（RA）患者的疾病活动相关。本研究旨在阐明其瓜氨酸化途径及其与中性粒细胞的相关性。通过免疫组织化学（IHC）、免疫沉淀（IP）和 Western blot（WB）检测 pGIA 关节中的 ITIH4 和 cit-ITIH4 水平，通过逆转录定量聚合酶链反应（RT-qPCR）、IHC 和免疫荧光方法检测肽基精氨酸脱亚氨酶（PAD）表达。pGIA 小鼠接受抗淋巴细胞抗原 6 复合物基因座 G6D（Ly6G）抗体以耗尽中性粒细胞，并通过 WB 研究 cit-ITIH4 的表达。通过 IP 和 WB 检测 RA 和骨关节炎（OA）患者滑液（SF）中的 ITIH4 和 cit-ITIH4 含量。在体外将重组 ITIH4 和 cit-ITIH4 与健康志愿者的血清孵育，然后使用 Boyden 室测定法和酶联免疫吸附测定（ELISA）评估其趋化能力和 C5a 水平。在关节炎高峰期，关节中 ITIH4 和 cit-ITIH4 增加，同时 PAD4 过表达，尤其是在 pGIA 小鼠浸润的中性粒细胞中。中性粒细胞耗竭后，血浆和关节中的 cit-ITIH4 水平显著降低。与 OA 患者相比，RA 患者 SF 中的 ITIH4 特异性瓜氨酸化。天然 ITIH4 抑制中性粒细胞迁移并降低 C5a 水平，而 cit-ITIH4 则显著增加其迁移和 C5a 水平。Cit-ITIH4 主要由中性粒细胞通过 PAD4 在炎症关节中产生。ITIH4 的瓜氨酸化可能通过激活补体级联反应改变其功能，从而上调中性粒细胞迁移，加重关节炎。
