Purpose: During a neuronal injury, a variety of immune cells infiltrate into the local microenvironment at the demyelination site. After the destruction of the intact myelin sheath, its major constituent myelin basic protein (MBP) dissociates from the plasma membrane and acts as a free ligand on the infiltrated immune cells. MBP exhibits charge microheterogeneity as a result of post-translational modifications, but the effect of various isomers of MBP on the activity of macrophages is not known.
Materials and methods: MBP was isolated and purified from bovine brain white matter. RAW 264.7 macrophages were cultured in DMEM supplemented with heat-inactivated fetal bovine serum. For evaluation of macrophage polarization following treatment of RAW 264.7 cells with MBP charge isomers, inducible nitric oxide synthase (iNOS) expression (M1 phenotype marker) and arginase-1 expression (M2 phenotype marker) were determined in cell lysates by ELISA. To assess Rac activity, G-LISA Rac Activation Assay system was used. The expression of receptor for advanced glycation end-products (RAGE) and high mobility group box 1 (HMGB1) protein were assayed by Western blot analysis.
Results: Our results have shown that minimally modified C1 component of MBP increases the expression of arginase-1 in cells, decreases the expression of iNOS, does not change the secretion of HMGB1 protein, but significantly elevates surface expression of RAGE, and in parallel, increases the activity of small GTPase Rac. On the other hand, highly modified deiminated isomer C8-MBP increases the secretion of HMGB1 protein but does not change the expression of arginase-1 or the content of RAGE.
Conclusion: These data indicate that deiminated C8 isomer of MBP tends to polarize RAW macrophages into M1 phenotypes, whereas C1 enhances the activity of M2 phenotype markers.

Keywords: Arginase-1, iNOS, HMGB1, RAGE




中文翻译：

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髓鞘碱性蛋白电荷异构体改变巨噬细胞极化。

目的：在神经元损伤期间，多种免疫细胞浸润到脱髓鞘部位的局部微环境中。在完整的髓鞘被破坏后，其主要成分髓鞘碱性蛋白 (MBP) 从质膜上解离出来，并在浸润的免疫细胞上充当游离配体。由于翻译后修饰，MBP表现出电荷微异质性，但MBP的各种异构体对巨噬细胞活性的影响尚不清楚。
材料和方法：MBP 是从牛脑白质中分离和纯化的。RAW 264.7 巨噬细胞在补充有热灭活胎牛血清的 DMEM 中培养。为了评估用 MBP 电荷异构体处理 RAW 264.7 细胞后的巨噬细胞极化，通过 ELISA 测定细胞裂解物中的诱导型一氧化氮合酶 (iNOS) 表达（M1 表型标记）和精氨酸酶-1 表达（M2 表型标记）。为了评估 Rac 活性，使用了 G-LISA Rac 激活测定系统。通过蛋白质印迹分析测定晚期糖基化终产物受体（RAGE）和高迁移率组框1（HMGB1）蛋白的表达。
结果：我们的研究结果表明，微量修饰的 MBP 的 C1 组分增加了细胞中 arginase-1 的表达，降低了 iNOS 的表达，不改变 HMGB1 蛋白的分泌，但显着提高了 RAGE 的表面表达，同时增加了 RAGE 的表面表达。小 GTPase Rac 的活性。另一方面，高度修饰的脱亚胺异构体 C8-MBP 增加了 HMGB1 蛋白的分泌，但不改变 arginase-1 的表达或 RAGE 的含量。
结论：这些数据表明 MBP 的脱亚胺 C8 异构体倾向于将 RAW 巨噬细胞极化为 M1 表型，而 C1 增强了 M2 表型标志物的活性。

关键词： Arginase-1，iNOS，HMGB1，RAGE