The Davis Heart and Lung Research Institute, The Ohio State University, Columbus, 43210, OH, USA.
J Inflamm (Lond). 2010 Jan 21;7(1):5. doi: 10.1186/1476-9255-7-5.
The granulocyte colony-stimulating factor receptor (G-CSFR) plays a critical role in maintaining homeostatic levels of circulating neutrophils (PMN). The mechanisms modulating G-CSFR surface expression to prevent chronic neutrophilia are poorly understood. Here, we report that neutrophil elastase (NE) proteolytically cleaves the G-CSFR on human PMN and blocks G-CSFR-mediated granulopoiesis in vitro.
Human peripheral blood PMN isolated from healthy donors were incubated with NE. Expression of the G-CSFR was analyzed by flow cytometry and western blot analyses. Detection of G-CSFR cleavage products from the culture supernatants was also performed. Human bone marrow mononuclear cells were also cultured in the presence or absence of NE to determine its effects on the proliferation of granulocyte-macrophage colony forming units (CFU-GM).
Treatment of PMN with NE induced a time-dependent decrease in G-CSFR expression that correlated with its degradation and the appearance of proteolytic cleavage fragments in conditioned media. Immunoblot analysis confirmed the G-CSFR was cleaved at its amino-terminus. Treatment of progenitor cells with NE prior to culture inhibited the growth of granulocyte-macrophage colony forming units.
These findings indicate that in addition to transcriptional controls and ligand-induced internalization, direct proteolytic cleavage of the G-CSFR by NE also downregulates G-CSFR expression and inhibits G-CSFR-mediated granulopoiesis in vitro. Our results suggest that NE negatively regulates granulopoiesis through a novel negative feedback loop.
粒细胞集落刺激因子受体（G-CSFR）在维持循环中性粒细胞（PMN）的稳态水平中起着关键作用。调节 G-CSFR 表面表达以防止慢性中性粒细胞增多的机制尚不清楚。在这里，我们报告中性粒细胞弹性蛋白酶（NE）可在体外对人 PMN 上的 G-CSFR 进行蛋白水解切割，并阻断 G-CSFR 介导的粒细胞生成。
从健康供体中分离出人外周血 PMN，并用 NE 孵育。通过流式细胞术和 Western blot 分析来分析 G-CSFR 的表达。还从培养上清液中检测 G-CSFR 切割产物。也在存在或不存在 NE 的情况下培养人骨髓单核细胞，以确定其对粒细胞-巨噬细胞集落形成单位（CFU-GM）增殖的影响。
PMN 用 NE 处理会导致 G-CSFR 表达的时间依赖性下降，这与 G-CSFR 的降解及其在条件培养基中出现的蛋白水解切割片段相关。免疫印迹分析证实 G-CSFR 在其氨基末端被切割。在培养前用 NE 处理祖细胞会抑制粒细胞-巨噬细胞集落形成单位的生长。
这些发现表明，除了转录控制和配体诱导的内化之外，NE 还通过直接蛋白水解切割 G-CSFR 来下调 G-CSFR 表达并抑制体外 G-CSFR 介导的粒细胞生成。我们的结果表明，NE 通过新的负反馈环负调节粒细胞生成。
