Department of Comparative Medicine, The Interuniversity Messerli Research Institute, University of Veterinary Medicine Vienna, Medical University of Vienna, University Vienna, Vienna, Austria.
Institute for Hygiene and Applied Immunology, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria.
Allergy. 2019 Mar;74(3):483-494. doi: 10.1111/all.13635. Epub 2018 Nov 28.
Macrophages can be converted in vitro into immunoregulatory M2b macrophages in the presence of immune complexes (ICs), but the role of the specific subclasses IgG1 or IgG4 in this phenotypic and functional change is not known.
We aimed to refine the original method by applying precisely defined ICs of the subclasses IgG4 or IgG1 constructed by two independent methods.
Monocyte-derived macrophages (MDMs) were treated with M-CSF, followed by IL-4/IL-13 to induce the M2a allergic phenotype. To mimic unspecific or allergen-specific ICs, plates were coated with myeloma IgG1 or IgG4, or with grass pollen allergen Phl p 5 followed by recombinant human Phl p 5-specific IgG1 or IgG4. M2a polarized macrophages were then added, cultured, and examined for cellular markers and cytokines by flow cytometry, ELISA, and rtPCR. Alternatively, immune complexes with IgG1 or IgG4 were formed using protein L.
IgG4 ICs down regulated CD163 and CD206 on M2a cells, and significantly increased IL-10, IL-6, TNFα, and CCL1 secretion, indicating a shift to an M2b-like phenotype. Treatment with IgG4 ICs resulted in expression of FcγRII and down modulation of FcγRII compared with IgG1 treated cells (P = 0.0335) or untreated cells (P < 0.00001).
Immune complexes with subclasses IgG1 and IgG4 can in vitro be generated by plate absorption, and in fluid form by protein L. Cross-linking of FcγRIIb by the IgG4 subclass redirects pro-allergic M2a macrophages to an M2b-like immunosuppressive phenotype. This suggests an interplay of macrophages with IgG4 in immune tolerance, likely relevant in allergen immunotherapy.
在免疫复合物（ICs）存在的情况下，巨噬细胞可以在体外转化为免疫调节型 M2b 巨噬细胞，但 IgG1 或 IgG4 亚类在这种表型和功能变化中的具体作用尚不清楚。
我们旨在通过应用两种独立方法构建的精确定义的 IgG4 或 IgG1 亚类免疫复合物，对原始方法进行改进。
单核细胞衍生的巨噬细胞（MDMs）用 M-CSF 处理，然后用 IL-4/IL-13 诱导 M2a 过敏表型。为了模拟非特异性或过敏原特异性 ICs，将板用骨髓瘤 IgG1 或 IgG4 或草花粉过敏原 Phl p 5 包被，然后用重组人 Phl p 5 特异性 IgG1 或 IgG4 包被。然后加入 M2a 极化的巨噬细胞，培养并通过流式细胞术、ELISA 和 rtPCR 检查细胞标记物和细胞因子。或者，使用蛋白 L 形成 IgG1 或 IgG4 的免疫复合物。
IgG4 IC 下调了 M2a 细胞上的 CD163 和 CD206，并显著增加了 IL-10、IL-6、TNFα 和 CCL1 的分泌，表明向 M2b 样表型转变。与 IgG1 处理的细胞（P=0.0335）或未处理的细胞（P<0.00001）相比，用 IgG4 IC 处理导致 FcγRII 的表达和 FcγRII 的下调。
通过平板吸收和蛋白 L 可以在体外生成 IgG1 和 IgG4 亚类的免疫复合物，通过 FcγRIIb 的交联将原过敏 M2a 巨噬细胞重定向为 M2b 样免疫抑制表型。这表明 IgG4 与巨噬细胞之间存在相互作用，可能与过敏原免疫治疗有关。
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