Department of Periodontology and Oral Biology, Goldman School of Dental Medicine, Boston University, Boston, MA 02118, USA.
J Immunol. 2010 Feb 1;184(3):1507-15. doi: 10.4049/jimmunol.0901219. Epub 2010 Jan 6.
Neutrophils from people with poorly controlled diabetes present a primed phenotype and secrete excessive superoxide. Phospholipase A(2) (PLA(2))-derived arachidonic acid (AA) activates the assembly of NADPH oxidase to generate superoxide anion. There is a gap in the current literature regarding which PLA(2) isoform regulates NADPH oxidase activation. The aim of this study was to identify the PLA(2) isoform involved in the regulation of superoxide generation in neutrophils and investigate if PLA(2) mediates priming in response to pathologic hyperglycemia. Neutrophils were isolated from people with diabetes mellitus and healthy controls, and HL60 neutrophil-like cells were grown in hyperglycemic conditions. Incubating neutrophils with the Ca(2+)-independent PLA(2) (iPLA(2)) inhibitor bromoenol lactone (BEL) completely suppressed fMLP-induced generation of superoxide. The nonspecific actions of BEL on phosphatidic acid phosphohydrolase-1, p47(phox) phosphorylation, and apoptosis were ruled out by specific assays. Small interfering RNA knockdown of iPLA(2) inhibited superoxide generation by neutrophils. Neutrophils from people with poorly controlled diabetes and in vitro incubation of neutrophils with high glucose and the receptor for advanced glycation end products ligand S100B greatly enhanced superoxide generation compared with controls, and this was significantly inhibited by BEL. A modified iPLA(2) assay, Western blotting, and PCR confirmed that there was increased iPLA(2) activity and expression in neutrophils from people with diabetes. AA (10 microM) partly rescued the inhibition of superoxide generation mediated by BEL, confirming that NADPH oxidase activity is, in part, regulated by AA. This study provides evidence for the role of iPLA(2) in enhanced superoxide generation in neutrophils from people with diabetes mellitus and presents an alternate pathway independent of protein kinase C and phosphatidic acid phosphohydrolase-1 hydrolase signaling.
来自血糖控制不佳的糖尿病患者的中性粒细胞呈现出预先激活的表型，并分泌过多的超氧阴离子。磷脂酶 A2（PLA2）衍生的花生四烯酸（AA）激活 NADPH 氧化酶的组装，产生超氧阴离子。目前关于哪种 PLA2 同工型调节 NADPH 氧化酶激活的文献存在空白。本研究旨在确定参与调节中性粒细胞中超氧阴离子生成的 PLA2 同工型，并研究 PLA2 是否介导病理高血糖引起的预激活。从糖尿病患者和健康对照者中分离中性粒细胞，并在高血糖条件下培养 HL60 中性粒细胞样细胞。用钙非依赖性 PLA2（iPLA2）抑制剂溴烯内酯（BEL）孵育中性粒细胞可完全抑制 fMLP 诱导的超氧阴离子生成。通过特异性测定排除了 BEL 对磷酸脂酸磷酸水解酶-1、p47（phox）磷酸化和细胞凋亡的非特异性作用。iPLA2 的小干扰 RNA 敲低抑制了中性粒细胞的超氧阴离子生成。与对照相比，血糖控制不佳的糖尿病患者的中性粒细胞和高糖与晚期糖基化终产物配体 S100B 的体外孵育大大增强了超氧阴离子的生成，BEL 显著抑制了这一作用。改良的 iPLA2 测定法、Western blot 和 PCR 证实糖尿病患者中性粒细胞中 iPLA2 活性和表达增加。10 μM AA 部分挽救了 BEL 介导的超氧阴离子生成抑制，证实 NADPH 氧化酶活性部分受 AA 调节。本研究为 iPLA2 在糖尿病患者中性粒细胞中超氧阴离子生成增强中的作用提供了证据，并提出了一种独立于蛋白激酶 C 和磷酸脂酸磷酸水解酶-1 水解酶信号的替代途径。
