Udsen Maja, Tagmose Christian, Garred Peter, Nissen Mogens Holst, Faber Carsten
Department of Immunology and Microbiology, University of Copenhagen, Faculty of Health and Medical Sciences, Copenhagen, Denmark.
Laboratory of Molecular Medicine, Department of Clinical Immunology, Section 7631, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark.
Exp Eye Res. 2022 May;218:108982. doi: 10.1016/j.exer.2022.108982. Epub 2022 Feb 17.
Age-related macular degeneration (AMD) has been associated with both complement activation and increased levels of circulating cytokines. Here, we sougth to investigate if cytokine-preexposure of retinal pigment epithelial (RPE) leads to increased complement activation and deposition of membrane attack complex (MAC). Primary human RPE and the ARPE19 cell line cultured in serum-free conditions were preexposed to 100 ng/ml interferon-gamma (IFNγ) and 20 ng/ml tumor necrosis factor-alpha (TNFα) for 48 h followed by exposure to diluted serum from healthy donors or complement factor B deficient (CFBd) serum for 70 min. Deposition of membrane attack complexes (MAC) was examined by use of a MAC-ELISA kit and by immunofluorescence. Eculizumab (anti-C5) was examined for its ability to prevent deposition of MAC on RPE cells exposed to serum. Lactatdehydrogenase (LDH) and thiazolyl blue tetrazolium bromide (MTT) assays were used to assess cellular metabolism and survival. MAC was deposited only on RPE preexposed to both IFNγ and TNFα. Lack of complement factor B or inhibition of C5 abrogated the MAC-deposition on RPE cells, while reconstitution of CFBd serum with CFB resulted in MAC-deposition. MAC-deposition resulted in RPE-release of LDH, but unaltered mitochondrial activity estimated by MTT. We conclude that preexposure of primary RPE and ARPE19 with inflammatory cytokines promoted alternative pathway activation of complement and deposition of MAC. This implies that circulating inflammatory mediators may increase susceptibility to local complement activation and MAC-deposition, which may represent an early event in the pathogenesis leading to AMD development.
年龄相关性黄斑变性（AMD）与补体激活和循环细胞因子水平升高均有关联。在此，我们旨在研究视网膜色素上皮（RPE）细胞预先暴露于细胞因子是否会导致补体激活增加以及膜攻击复合物（MAC）沉积。在无血清条件下培养的原代人RPE细胞和ARPE19细胞系预先暴露于100 ng/ml干扰素-γ（IFNγ）和20 ng/ml肿瘤坏死因子-α（TNFα）48小时，随后暴露于来自健康供体的稀释血清或补体因子B缺陷（CFBd）血清70分钟。通过使用MAC-ELISA试剂盒和免疫荧光检查膜攻击复合物（MAC）的沉积情况。研究了依库珠单抗（抗C5）预防MAC在暴露于血清的RPE细胞上沉积的能力。使用乳酸脱氢酶（LDH）和噻唑蓝四氮唑溴盐（MTT）测定法评估细胞代谢和存活率。MAC仅沉积在预先暴露于IFNγ和TNFα的RPE细胞上。缺乏补体因子B或抑制C5可消除RPE细胞上的MAC沉积，而用CFB重建CFBd血清则导致MAC沉积。MAC沉积导致RPE释放LDH，但MTT估计的线粒体活性未改变。我们得出结论，原代RPE细胞和ARPE19细胞预先暴露于炎性细胞因子会促进补体的替代途径激活和MAC沉积。这意味着循环炎性介质可能会增加局部补体激活和MAC沉积的易感性，这可能是导致AMD发生的发病机制中的早期事件。
