Abstract：
OBJECTIVE: Neutrophils perform various functions in a circadian-dependent manner; therefore, we investigated here whether the effect of alpha1-antitrypsin (AAT), used as augmentation therapy, is dependent on the neutrophil circadian clock. AAT is a vital regulator of neutrophil functions, and its qualitative and/or quantitative defects have significant implications for the development of respiratory diseases.
METHODS: Whole blood from 12 healthy women age years, mean (SD) 29.92 (5.48) was collected twice daily, 8 h apart, and incubated for 30 min at 37 °C alone or with additions of 2 mg/ml AAT (Respreeza) and/or 5 μg/ml lipopolysaccharide (LPS) from Escherichia coli. Neutrophils were then isolated to examine gene expression, migration and phagocytosis.
RESULTS: The expression of CD14, CD16, CXCR2 and SELL (encoding CD62L) genes was significantly higher while CDKN1A lower in the afternoon than in the morning neutrophils from untreated blood. Neutrophils isolated in the afternoon had higher migratory and phagocytic activity. Morning neutrophils isolated from AAT-pretreated blood showed higher expression of CXCR2 and SELL than those from untreated morning blood. Pretreatment of blood with AAT enhanced migratory properties of morning but not afternoon neutrophils. Of all genes analysed, only CXCL8 expression was strongly upregulated in morning and afternoon neutrophils isolated from LPS-pretreated blood, whereas CXCR2 expression was downregulated in afternoon neutrophils. The addition of AAT did not reverse the effects of LPS.
CONCLUSIONS: The circadian clock of myeloid cells may affect the effectiveness of various therapies, including AAT therapy used to treat patients with AAT deficiency, and needs further investigation.
METHODS: Whole blood from 12 healthy women age years, mean (SD) 29.92 (5.48) was collected twice daily, 8 h apart, and incubated for 30 min at 37 °C alone or with additions of 2 mg/ml AAT (Respreeza) and/or 5 μg/ml lipopolysaccharide (LPS) from Escherichia coli. Neutrophils were then isolated to examine gene expression, migration and phagocytosis.
RESULTS: The expression of CD14, CD16, CXCR2 and SELL (encoding CD62L) genes was significantly higher while CDKN1A lower in the afternoon than in the morning neutrophils from untreated blood. Neutrophils isolated in the afternoon had higher migratory and phagocytic activity. Morning neutrophils isolated from AAT-pretreated blood showed higher expression of CXCR2 and SELL than those from untreated morning blood. Pretreatment of blood with AAT enhanced migratory properties of morning but not afternoon neutrophils. Of all genes analysed, only CXCL8 expression was strongly upregulated in morning and afternoon neutrophils isolated from LPS-pretreated blood, whereas CXCR2 expression was downregulated in afternoon neutrophils. The addition of AAT did not reverse the effects of LPS.
CONCLUSIONS: The circadian clock of myeloid cells may affect the effectiveness of various therapies, including AAT therapy used to treat patients with AAT deficiency, and needs further investigation.
摘要：
目的：中性粒细胞以昼夜节律依赖的方式执行各种功能；因此，我们在这里研究了α1-抗胰蛋白酶(AAT)的作用，用作增强疗法，取决于中性粒细胞的昼夜节律。AAT是中性粒细胞功能的重要调节因子,及其定性和/或定量缺陷对呼吸系统疾病的发展具有重要意义。
方法：来自12名健康女性的全血[年龄，平均（SD）29.92（5.48）每天两次收集，相隔8小时，并在37°C下单独或添加来自大肠杆菌的2mg/mlAAT（Respreeza）和/或5μg/ml脂多糖（LPS）孵育30分钟。然后分离中性粒细胞以检查基因表达，迁移和吞噬作用。
结果：与未处理血液中的早晨嗜中性粒细胞相比，下午CD14，CD16，CXCR2和SELL（编码CD62L）基因的表达明显更高，而CDKN1A则更低。下午分离的中性粒细胞具有较高的迁移和吞噬活性。从AAT预处理的血液中分离出的早晨嗜中性粒细胞显示出比未处理的早晨血液中更高的CXCR2和SELL表达。用AAT预处理血液可增强早晨但不增强下午中性粒细胞的迁移特性。在所有被分析的基因中，仅CXCL8表达在早晨和下午从LPS预处理的血液中分离的中性粒细胞强烈上调，而CXCR2表达在下午中性粒细胞中下调。AAT的添加没有逆转LPS的作用。
结论：骨髓细胞的生物钟可能会影响各种疗法的有效性，包括用于治疗AAT缺乏症患者的AAT疗法，需要进一步调查.
方法：来自12名健康女性的全血[年龄，平均（SD）29.92（5.48）每天两次收集，相隔8小时，并在37°C下单独或添加来自大肠杆菌的2mg/mlAAT（Respreeza）和/或5μg/ml脂多糖（LPS）孵育30分钟。然后分离中性粒细胞以检查基因表达，迁移和吞噬作用。
结果：与未处理血液中的早晨嗜中性粒细胞相比，下午CD14，CD16，CXCR2和SELL（编码CD62L）基因的表达明显更高，而CDKN1A则更低。下午分离的中性粒细胞具有较高的迁移和吞噬活性。从AAT预处理的血液中分离出的早晨嗜中性粒细胞显示出比未处理的早晨血液中更高的CXCR2和SELL表达。用AAT预处理血液可增强早晨但不增强下午中性粒细胞的迁移特性。在所有被分析的基因中，仅CXCL8表达在早晨和下午从LPS预处理的血液中分离的中性粒细胞强烈上调，而CXCR2表达在下午中性粒细胞中下调。AAT的添加没有逆转LPS的作用。
结论：骨髓细胞的生物钟可能会影响各种疗法的有效性，包括用于治疗AAT缺乏症患者的AAT疗法，需要进一步调查.
