New model of macrophage acquisition of the lymphatic endothelial phenotype.
New model of macrophage acquisition of the lymphatic endothelial phenotype.
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DOI：
10.1371/journal.pone.0031794
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Ran S
中科院分区：
文献类型：
--
作者：
Hall KL;Volk-Draper LD;Flister MJ;Ran S
巨噬细胞来源的淋巴管内皮祖细胞（M-LECPs）有助于新的淋巴管形成，但调节其分化，招募和功能的机制知之甚少。M-LECPs的详细表征受到体内低频率和缺乏允许体外深入分子分析的模型系统的限制。我们的目标是建立一个细胞培养模型，以表征在受控条件下炎症诱导的巨噬细胞向LECP分化。从脂多糖（LPS）处理的小鼠横膈膜的时间过程分析显示，骨髓来源的和腹膜巨噬细胞的淋巴管附近的迅速动员，随后广泛（约50%）的M-LECP纳入发炎的淋巴管。在三个LPS诱导的活化巨噬细胞亚群中发现了向淋巴细胞表型的分化转变，这些亚群对VEGFR-3和许多其他淋巴细胞特异性标志物呈阳性。VEGFR-3在巨噬细胞向LECP转化的早期阶段强烈升高，但在血管整合之前在M-LECP中检测不到。在体外LPS激活的RAW264.7巨噬细胞中发现了类似的VEGFR-3瞬时表达模式。活化的RAW264.7细胞共表达诱导自分泌信号环的VEGF-C，如可溶性受体抑制的VEGFR-3磷酸化所示。在淋巴特异性基因的表达方面，LPS激活的RAW264.7巨噬细胞也与内源性CD 11b +/VEGFR-3+ LECP显示出68%的重叠。此外，当注射到LPS而不是盐水处理的小鼠中时，GFP标记的RAW 264.7细胞大量浸润发炎的隔膜，随后整合到18%的淋巴管中。我们提出了一种新的模型，巨噬细胞LECP分化的基础上培养的RAW 264.7细胞的LPS激活。该系统在这里指定为“RAW模型”模仿内源性M-LECP的基本特征。与天然LECP不同，该模型不受细胞数量、群体异质性和出于实验目的改变遗传组成的能力的限制。因此，该模型可以为理解LECP和淋巴生物学提供有价值的工具。
Macrophage-derived lymphatic endothelial cell progenitors (M-LECPs) contribute to new lymphatic vessel formation, but the mechanisms regulating their differentiation, recruitment, and function are poorly understood. Detailed characterization of M-LECPs is limited by low frequency in vivo and lack of model systems allowing in-depth molecular analyses in vitro. Our goal was to establish a cell culture model to characterize inflammation-induced macrophage-to-LECP differentiation under controlled conditions. Time-course analysis of diaphragms from lipopolysaccharide (LPS)-treated mice revealed rapid mobilization of bone marrow-derived and peritoneal macrophages to the proximity of lymphatic vessels followed by widespread (∼50%) incorporation of M-LECPs into the inflamed lymphatic vasculature. A differentiation shift toward the lymphatic phenotype was found in three LPS-induced subsets of activated macrophages that were positive for VEGFR-3 and many other lymphatic-specific markers. VEGFR-3 was strongly elevated in the early stage of macrophage transition to LECPs but undetectable in M-LECPs prior to vascular integration. Similar transient pattern of VEGFR-3 expression was found in RAW264.7 macrophages activated by LPS in vitro. Activated RAW264.7 cells co-expressed VEGF-C that induced an autocrine signaling loop as indicated by VEGFR-3 phosphorylation inhibited by a soluble receptor. LPS-activated RAW264.7 macrophages also showed a 68% overlap with endogenous CD11b+/VEGFR-3+ LECPs in the expression of lymphatic-specific genes. Moreover, when injected into LPS- but not saline-treated mice, GFP-tagged RAW264.7 cells massively infiltrated the inflamed diaphragm followed by integration into 18% of lymphatic vessels. We present a new model for macrophage-LECP differentiation based on LPS activation of cultured RAW264.7 cells. This system designated here as the “RAW model” mimics fundamental features of endogenous M-LECPs. Unlike native LECPs, this model is unrestricted by cell numbers, heterogeneity of population, and ability to change genetic composition for experimental purposes. As such, this model can provide a valuable tool for understanding the LECP and lymphatic biology.
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3.7
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