title: Diabetes-induced oxidative stress is mediated by Ca2+-independent phospholipase A2 in neutrophils.
abstract: 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.
authors.affiliation: Department of Periodontology and Oral Biology, Goldman School of Dental Medicine, Boston University, Boston, MA 02118, USA.
authors.forename: Srinivas
authors.initials: S
authors.lastname: Ayilavarapu
authors.forename: Alpdogan
authors.initials: A
authors.lastname: Kantarci
authors.forename: Gabrielle
authors.initials: G
authors.lastname: Fredman
authors.forename: Oya
authors.initials: O
authors.lastname: Turkoglu
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Omori
authors.forename: Hongsheng
authors.initials: H
authors.lastname: Liu
authors.forename: Tomoyuki
authors.initials: T
authors.lastname: Iwata
authors.forename: Motohiko
authors.initials: M
authors.lastname: Yagi
authors.forename: Hatice
authors.initials: H
authors.lastname: Hasturk
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Van Dyke
mesh_terms: D000328:Adult; D002118:Calcium / Q000097:blood* / Q000502:physiology; D003922:Diabetes Mellitus, Type 1 / Q000201:enzymology / Q000276:immunology* / Q000378:metabolism*; D003924:Diabetes Mellitus, Type 2 / Q000201:enzymology / Q000276:immunology* / Q000378:metabolism*; D005260:Female; D018390:Gene Targeting; D054522:Group VI Phospholipases A2 / Q000037:antagonists & inhibitors / Q000097:blood* / Q000235:genetics / Q000502:physiology; D018922:HL-60 Cells; D006801:Humans; D008297:Male; D008875:Middle Aged; D009504:Neutrophils / Q000201:enzymology* / Q000276:immunology; D018384:Oxidative Stress / Q000276:immunology*; D010711:Phosphatidate Phosphatase / Q000502:physiology; D011493:Protein Kinase C / Q000502:physiology; D015398:Signal Transduction / Q000235:genetics / Q000276:immunology; D013481:Superoxides / Q000097:blood; D015854:Up-Regulation / Q000276:immunology
chemical_list: D013481:Superoxides; D011493:Protein Kinase C; D054522:Group VI Phospholipases A2; C516475:PLA2G6 protein, human; D010711:Phosphatidate Phosphatase; D002118:Calcium