Macrophages are professional phagocytes that are uniquely situated between the innate and adaptive arms of immunity with a high capacity for phagocytosis and proinflammatory cytokine production as well as antigen presentation. Phagocytosis is a critical process to eliminate microbes, apoptotic cells and other foreign particles and is accelerated by host-generated opsonins, such as antibodies and complement. Early phagocytosis studies established the paradigm that FcγR-mediated phagocytosis was more proinflammatory than Complement Receptor (CR)-mediated uptake in macrophages. Using qPCR, cytokine antibody arrays and ELISA, we revisited this research question in primary macrophages. Using qPCR we determined that CR-mediated phagocytosis increases levels of TNF-α, IL-1β, IL-6, and MMP-9, compared to FcγR-mediated phagocytosis and control unstimulated cells. We confirmed these findings at the protein level using cytokine antibody arrays and ELISAs. We next investigated the mechanism behind upregulated cytokine production during CR-mediated phagocytosis. IκBα protein levels were reduced after phagocytosis of both IgG- and C3bi-sRBCs indicating proteolytic degradation and implicating NF-κB activation. Inhibition of NF-κB activation impacted IL-6 production during phagocytosis in macrophages. Due to the roles of calpain in IκBα and integrin degradation, we hypothesized that CR-mediated phagocytosis may utilize calpain for proinflammatory mediator enhancement. Using qPCR and cytokine antibody array analysis, we saw significant reduction of cytokine expression during CR-mediated phagocytosis following the addition of the calpain inhibitor, PD150606, compared to untreated cells. These results suggest that the upregulation of proinflammatory mediators during CR-mediated phagocytosis is potentially dependent upon calpain-mediated activation of NF-κB.

中文翻译：

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补体受体介导的吞噬作用诱导小鼠巨噬细胞中促炎性细胞因子的产生。

巨噬细胞是专业的吞噬细胞，其独特地位于先天免疫和适应性免疫之间，具有吞噬作用和促炎细胞因子产生以及抗原呈递的高容量。吞噬作用是消除微生物，凋亡细胞和其他外来颗粒的关键过程，并由宿主产生的调理素（例如抗体和补体）加速。早期吞噬作用研究确立了范式，即在巨噬细胞中，FcγR介导的吞噬作用比补体受体（CR）介导的摄取更具促炎性。使用qPCR，细胞因子抗体阵列和ELISA，我们在原代巨噬细胞中重新研究了该研究问题。通过qPCR，我们确定了CR介导的吞噬作用可增加TNF-α，IL-1β，IL-6和MMP-9的水平，与FcγR介导的吞噬作用和对照未刺激细胞相比。我们使用细胞因子抗体阵列和ELISA在蛋白质水平上证实了这些发现。接下来，我们研究了CR介导的吞噬作用过程中细胞因子产生上调的机制。吞噬IgG-和C3bi-sRBC后，IκBα蛋白水平降低，表明蛋白水解降解并暗示NF-κB活化。巨噬细胞吞噬过程中抑制NF-κB活化会影响IL-6的产生。由于钙蛋白酶在IκBα和整合素降解中的作用，我们假设CR介导的吞噬作用可能利用钙蛋白酶促进促炎性介质的表达。使用qPCR和细胞因子抗体阵列分析，我们发现与未处理的细胞相比，加入钙蛋白酶抑制剂PD150606后，CR介导的吞噬作用期间细胞因子表达显着降低。这些结果表明在CR介导的吞噬过程中促炎介质的上调可能取决于钙蛋白酶介导的NF-κB的活化。