Tumor necrosis factor alpha derived from classically activated "M1" macrophages reduces interstitial cell of Cajal numbers.
Tumor necrosis factor alpha derived from classically activated "M1" macrophages reduces interstitial cell of Cajal numbers.
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DOI：
10.1111/nmo.12984
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发表时间：
2017-04
影响因子：
3.5
通讯作者：
Farrugia G
中科院分区：
文献类型：
--
作者：
Eisenman ST;Gibbons SJ;Verhulst PJ;Cipriani G;Saur D;Farrugia G
糖尿病小鼠和人类的胃排空延迟与巨噬细胞表型的变化和胃肌层 Cajal 间质细胞 (ICC) 的损失有关。在糖尿病小鼠中，经典激活的 M1 巨噬细胞与胃排空延迟相关，而替代激活的 M2 巨噬细胞与正常胃排空相关。本研究旨在确定 M1 巨噬细胞分泌的因子是否会损伤原代培养的小鼠 ICC。用来自活化的骨髓源性巨噬细胞 (BMDM) 的条件培养基 (CM) 处理胃 ICC 培养物，并通过计算每个高倍视野的 ICC 来量化 CM 的效果。 BMDM 被激活为 M1 或 M2 表型，经 qRT-PCR 确认。来自 M1 巨噬细胞的 CM 使 ICC 数量减少了 41.1%，而与未条件的对照培养基相比，M2-CM 没有效果。对 40 种趋化因子/细胞因子的免疫印迹分析发现，其中 12 种在 M1-CM 中显着增加，包括肿瘤坏死因子 α (TNFα)。 ELISA 在 M1-CM 中检测到 0.697 ± 0.03 ng mL−1 TNFα。重组小鼠 TNFα 以浓度依赖性方式降低 Kit 表达和 ICC 数量（EC50 = 0.817 ng mL−1）。用中和抗体阻断 M1-CM TNFα 可以保留 ICC 值。 Caspase 抑制剂 Z-VAD.fmk 部分保留了 ICC 数量（细胞/视野；6.63±1.04、9.82±1.80 w/Z-VAD.fmk，n=6，P < 0.05）。这项工作表明，M1 巨噬细胞分泌的 TNFα 可导致 Kit 丢失，并部分通过 caspase 依赖性细胞凋亡直接损伤体外 ICC，并且可能在糖尿病胃轻瘫的 ICC 耗竭中发挥重要作用。在糖尿病小鼠中，M1 巨噬细胞与胃排空延迟相关，而 M2 巨噬细胞与正常胃排空相关。 TNFα 是 M1 条件培养基中存在的一种因子，可减少 Kit 阳性 ICC 数量，并且 TNFα 中和抗体可阻断 M1 培养基的作用。来自 M1 巨噬细胞的 TNFα 在体外会损伤 ICC，并且 TNFα 可能在糖尿病性胃轻瘫等疾病中发挥重要作用。
Delayed gastric emptying in diabetic mice and humans is associated with changes in macrophage phenotype and loss of interstitial cells of Cajal (ICC) in the gastric muscle layers. In diabetic mice, classically activated M1 macrophages are associated with delayed gastric emptying whereas alternatively activated M2 macrophages are associated with normal gastric emptying. This study aimed to determine if secreted factors from M1 macrophages could injure mouse ICC in primary culture. Cultures of gastric ICC were treated with conditioned medium (CM) from activated bone marrow-derived macrophages (BMDMs) and the effect of CM was quantified by counting ICC per high-powered field. BMDMs were activated to a M1 or M2 phenotype confirmed by qRT-PCR. CM from M1 macrophages reduced ICC numbers by 41.1%, while M2-CM had no effect as compared to unconditioned, control media. Immunoblot analysis of 40 chemokines/cytokines found 12 were significantly increased in M1-CM, including tumor necrosis factor alpha (TNFα). ELISA detected 0.697 ± 0.03 ng mL−1 TNFα in M1-CM. Recombinant mouse TNFα reduced Kit expression and ICC numbers in a concentration-dependent manner (EC50 = 0.817 ng mL−1). Blocking M1-CM TNFα with a neutralizing antibody preserved ICC numbers. The caspase inhibitor Z-VAD.fmk partly preserved ICC numbers (cells/field; 6.63±1.04, 9.82±1.80 w/Z-VAD.fmk, n=6, P < 0.05). This work demonstrates that TNFα secreted from M1 macrophages can result in Kit loss and directly injure ICC in vitro partly through caspase-dependent apoptosis and may play an important role in ICC depletion in diabetic gastroparesis. In diabetic mice, M1 macrophages are associated with delayed gastric emptying whereas M2 macrophages are associated with normal gastric emptying. TNFα, a factor present in M1 conditioned medium, reduced Kit-positive ICC numbers and TNFα neutralizing antibodies blocked the effect of M1 medium. TNFα derived from M1 macrophages injures ICC in vitro and TNFα may be important in diseases like diabetic gastroparesis.