A novel flow cytometric assay of human whole blood neutrophil and monocyte CD11b levels: Upregulation by chemokines is related to receptor expression, comparison with neutrophil shape change, and effects of a chemokine receptor (CXCR2) antagonist
A novel flow cytometric assay of human whole blood neutrophil and monocyte CD11b levels: Upregulation by chemokines is related to receptor expression, comparison with neutrophil shape change, and effects of a chemokine receptor (CXCR2) antagonist
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DOI：
10.1016/j.pupt.2005.11.009
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发表时间：
2007-01-01
影响因子：
3.2
通讯作者：
Hansel, Trevor T.
中科院分区：
文献类型：
--
作者：
Nicholson, Grant C.;Tennant, Rachel C.;Hansel, Trevor T.
理由：患慢性阻塞性肺病 (COPD) 的吸烟者肺部炎症加剧，涉及中性粒细胞、巨噬细胞和 CD8+ T 细胞的选择性组织积聚。 CD11b（Mac-1，α M β（2）-整合素）既是补体受体（CR3），也是存在于外周血白细胞表面的细胞粘附分子，并且在激活时从预先形成的细胞质储存中经历快速的表面上调。细胞激活还可以触发趋化性和形状变化，激活本身是由趋化因子与细胞表面受体的结合引起的。方法：我们开发了一种全血流式细胞术方法，在流式细胞术之前使用核染料 LDS-751 染色来测量 CD16+ 和 CD14+ 细胞上的中性粒细胞和单核细胞 CD11b 上调。此外，我们通过改良门控自发荧光前向散射 (GAFS) 评估中性粒细胞形状变化，这与趋化反应相关。 结果：与健康吸烟者相比，患有 COPD 的吸烟者中，中性粒细胞对 CXCL8 (IL-8) 的最大形状变化较低 (p = 0.025)，并且 CD11b 表达和形状变化对 CXCL I (GRO-alpha) 的反应较低。研究发现，中性粒细胞主要表达趋化因子受体 CXCR1 和 CXCR2，并对 CXCL8 做出反应，CD11b 上调，而单核细胞表达更多的 CCR2，并且优先于 CCL2 (MCP-1) 上调 CD11b。研究发现，CXCR2 拮抗剂 (SB-656933) 可抑制 COPD 患者中性粒细胞 CD11b 上调 (IC50 = 260.7 nM) 和形状改变 (IC50 = 310.5 nM)。结论：中性粒细胞和单核细胞参与一系列疾病的炎症过程。这些全血测定可用于监测疾病活动并对趋化因子受体 (CXCR) 拮抗剂进行体外和离体评估。 (c) 2005 Elsevier Ltd. 保留所有权利。
Rationale: Smokers who develop chronic obstructive pulmonary disease (COPD) have amplified inflammation within their lungs, involving selective tissue accumulation of neutrophils, macrophages and CD8+ T cells. CD11b (Mac-1, alpha M beta(2)-integrin) is both a complement receptor (CR3) and a cell adhesion molecule present on the surface of peripheral blood leukocytes, and undergoes rapid surface upregulation from preformed cytoplasmic stores on activation. Cellular activation can also trigger chemotaxis and shape change, the activation itself being caused by the binding of chemokines to cell surface receptors.Methods: We developed a method of whole blood flow cytometry to measure neutrophil and monocyte CD11b upregulation on CD16+ and CD14+ cells, employing staining with the nuclear dye LDS-751 immediately before flow cytometry. In addition we assessed neutrophil shape change by modified gated autofluorescence with forward scatter (GAFS), this being correlated with chemotactic responses.Results: In smokers with COPD there was a lower maximal shape change for neutrophils in response to CXCL8 (IL-8) in comparison to healthy smokers (p = 0.025), and a trend for lower expression of CD11b and shape change in response to CXCL I (GRO-alpha). Neutrophils were found to predominantly express chemokine receptors CXCR1 and CXCR2 and respond to CXCL8 with CD11b upregulation, while monocytes express more CCR2 and upregulate CD11b preferentially to CCL2 (MCP-1). A CXCR2 antagonist (SB-656933) was found to inhibit neutrophil CD11b upregulation (IC50 = 260.7 nM) and shape change (IC50 = 310.5 nM) in COPD patients.Conclusions: Neutrophils and monocytes participate in inflammatory processes in a range of diseases. These whole blood assays can be employed to monitor activity in disease and perform in vitro and ex vivo assessment of chemokine receptor (CXCR) antagonists. (c) 2005 Elsevier Ltd. All rights reserved.