流式细胞术
全血
CD63
N-甲酰甲硫氨酸亮氨酸苯丙氨酸
粒细胞
甲酰肽受体
兴奋剂
整合素αM
EC50型
血细胞
分子生物学
受体
中性粒细胞
免疫学
生物
受体表达
化学
趋化性
炎症
体外
生物化学
微泡
基因
小RNA
作者
Carola Ledderose,Naoyuki Hashiguchi,Eleftheria‐Angeliki Valsami,Christian Rusu,Wolfgang G. Junger
标识
DOI:10.1016/j.jim.2022.113403
摘要
Polymorphonuclear neutrophils (PMNs) protect the host from invading microorganisms. However, excessively activated PMNs can also cause damage to host tissues under inflammatory conditions. Here we developed simple assays to determine the activation state of PMNs in human whole blood that contains soluble mediators known to influence PMN functions. Because mouse models are widely used to study the role of PMNs in infectious and inflammatory diseases, we adapted these assays for the rapid and reliable assessment of PMN functions in murine blood samples. Freshly collected whole blood samples were stimulated with agonists of the formyl peptide receptors (FPR) of PMNs and changes in reactive oxygen species (ROS) production and the expression of CD11b, CD62L (L-selectin), CD66b, and CD63 on the cell surface were analyzed with flow cytometry. We optimized these assays to minimize inadvertent interferences such as cell stress generated during sample handling and the loss of plasma mediators that regulate PMN functions. Human PMNs readily responded to the FPR agonist N-formyl-methionyl-leucyl-phenylalanine (fMLP). The most sensitive responses of human PMNs to fMLP were CD11b, CD62L, and CD66b expression with half maximal effective concentrations (EC50) of 5, 8, and 6 nM fMLP, respectively. CD63 expression and ROS production required markedly higher fMLP concentrations with EC50 values of 19 and 50 nM fMLP, respectively. Mouse PMNs did not respond well to fMLP and required significantly higher concentrations of the FPR agonist WKYMVm (W-peptide) to achieve equivalent cell activation. The most sensitive response of mouse PMNs was ROS production with an EC50 of 38 nM W-peptide. Because mice do not express CD66b, we only assessed the expression of CD62L, CD11b, and CD63 with EC50 values of 54, 119, and 355 nM W-peptide, respectively. Validation of our optimized assays showed that they sensitively detect the responses of human PMNs to priming with endotoxin in vitro as well as the corresponding responses of murine PMNs to bacterial infection in a sepsis model. We conclude that these optimized assays could be useful tools for the monitoring of patients with infections, sepsis, and other inflammatory conditions as well as for the design and interpretation of preclinical studies of these diseases in mouse models.
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