Supplementary Material for: The Anaphylatoxin C3a Receptor Expression on Human M2 Macrophages Is Down-Regulated by Stimulating the Histamine H4 Receptor and the IL-4 Receptor

The anaphylatoxin C3a triggers inflammation by binding to its specific G-protein-coupled C3a receptor (C3aR). Since the number of C3aR, which is expressed on the cell surface, affects the response to C3a, we investigated the expression levels of C3aR on human M2 macrophages in allergic situations where high levels of the Th2 cytokine IL-4 and histamine are present in a local microenvironment. The histamine H1 receptor (H1R), H2R and the H4R mRNA expressions were induced or up-regulated during the differentiation process of M2 macrophages. The presence of histamine or agonists targeting the H1R, H2R and, in particular, the H4R during in vitro differentiation from monocytes to macrophages modified the M2 phenotype by regulating the macrophage differentiation marker CD68 and CD163 expressions. In ­addition, the C3aR expression was also down-regulated by ­ST-1006 during this process. Histamine and ST-1006 down-regulated the expression of C3aR with different time kinetics on fully differentiated M2 macrophages. By analysing C3a-induced IL-6 mRNA expression, we observed a diminished response to C3a in ST-1006-treated M2 macrophages when compared to un-treated cells. Expression of C3 was not affected by histamine, whereas IL-4 strongly down-regulated C3aR and C3 expressions. Our data suggests that down-regulation of C3aR expression by mediators present in allergic situations such as IL-4 or histamine has an anti-inflammatory impact by reducing the sensitivity to C3a-induced down-stream signaling, thereby contributing to the regulation of local inflammatory responses in the skin.

过敏毒素C3a（anaphylatoxin C3a）通过结合其特异性G蛋白偶联C3a受体（G-protein-coupled C3a receptor, C3aR）触发炎症反应。由于细胞表面表达的C3aR数量会影响细胞对C3a的应答能力，我们针对局部微环境中存在高浓度Th2细胞因子白介素4（interleukin-4, IL-4）与组胺的过敏病理状态，探究了人源M2巨噬细胞表面C3aR的表达水平。在单核细胞向巨噬细胞的体外分化过程中，组胺H1受体（histamine H1 receptor, H1R）、H2R及H4R的mRNA表达均被诱导或上调。若单核细胞向巨噬细胞体外分化阶段暴露于组胺，或靶向H1R、H2R（尤其是H4R）的激动剂，可通过调控巨噬细胞分化标志物CD68与CD163的表达，改变M2巨噬细胞表型。此外，在此分化过程中，ST-1006亦可下调C3aR的表达。组胺与ST-1006可通过不同的时间动力学特征，在完全分化的M2巨噬细胞中下调C3aR的表达。通过分析C3a诱导的白介素6（interleukin-6, IL-6）mRNA表达，我们观察到：与未处理组细胞相比，经ST-1006处理的M2巨噬细胞对C3a的应答显著减弱。组胺对补体C3（complement C3, C3）的表达无显著影响，而IL-4可强烈下调C3aR与C3的表达。本研究数据表明，过敏状态下存在的介质（如IL-4或组胺）可通过下调C3aR的表达，降低细胞对C3a诱导的下游信号通路的敏感性，从而发挥抗炎作用，参与调控皮肤局部的炎症反应。