一种检测人全血中性粒细胞和单核细胞CD11b水平的新型流式细胞术检测方法：趋化因子诱导的上调与受体表达有关，与中性粒细胞形态变化的比较，以及一种趋化因子受体（CXCR2）拮抗剂的作用
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.
作者信息
Nicholson Grant C, Tennant Rachel C, Carpenter Donald C, Sarau Henry M, Kon Onn Min, Barnes Peter J, Salmon Michael, Vessey Rupert S, Tal-Singer Ruth, Hansel Trevor T
机构信息
National Heart and Lung Institute (NHLI), Clinical Studies Unit, Imperial College London, Royal Brompton Hospital, Fulham Road, London SW3 6HP, UK.
出版信息
Pulm Pharmacol Ther. 2007;20(1):52-9. doi: 10.1016/j.pupt.2005.11.009. Epub 2006 Jan 6.
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, alphaMbeta(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 CXCL1 (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=260.7nM) and shape change (IC50=310.5nM) 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.
原理
患慢性阻塞性肺疾病（COPD）的吸烟者肺部炎症加剧，伴有中性粒细胞、巨噬细胞和CD8 + T细胞在组织中的选择性积聚。CD11b（Mac-1，αMβ2整合素）既是补体受体（CR3），也是外周血白细胞表面存在的细胞粘附分子，在激活时可从预先形成的细胞质储存中快速上调至细胞表面。细胞激活还可触发趋化性和形态变化，而激活本身是由趋化因子与细胞表面受体结合引起的。
方法
我们开发了一种全血流式细胞术方法，通过在流式细胞术检测前立即用核染料LDS-751染色，来测量CD16 +和CD14 +细胞上中性粒细胞和单核细胞CD11b的上调情况。此外，我们通过改良的前向散射门控自发荧光（GAFS）评估中性粒细胞的形态变化，这与趋化反应相关。
结果
与健康吸烟者相比，COPD吸烟者的中性粒细胞对CXCL8（IL-8）的最大形态变化较低（p = 0.025），并且对CXCL1（GRO-α）的CD11b表达和形态变化有降低的趋势。发现中性粒细胞主要表达趋化因子受体CXCR1和CXCR2，并通过CD11b上调对CXCL8作出反应，而单核细胞表达更多的CCR2，并优先对CCL2（MCP-1）上调CD11b。发现CXCR2拮抗剂（SB-656933）可抑制COPD患者中性粒细胞CD11b的上调（IC50 = 260.7nM）和形态变化（IC50 = 310.5nM）。
结论
中性粒细胞和单核细胞参与多种疾病的炎症过程。这些全血检测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。
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