Augmentation of the Neutrophil Respiratory Burst Through the Action of Advanced Glycation End Products

Abstract

An accelerated accumulation of advanced glycation end products (AGEs) occurs in diabetes secondary to the increased glycemic burden. In this study, we investigated the contribution of AGEs to intravascular oxidant stress by examining their action on the neutrophil burst of reactive oxygen species (ROS); this may be a significant donor to the overall vascular redox status and to vasculopathy. AGEs exerted a dose-dependent enhancement on the neutrophil respiratory burst in response to a secondary mechanical stimulus (up to 265 ؎ 42%, P ‫؍‬ 0.022) or chemical stimulation with formyl-methylleucylphenylalanine 100 nmol/l (up to 218 ؎ 19%, P < 0.001), although they possessed no ability to augment the neutrophil respiratory burst alone. This phenomenon was both immediate and reversible and depended on the simultaneous presence of AGEs with the additional stimulus. It appeared to work through an upregulation of the neutrophil NADPH oxidase, the enzyme responsible for ROS generation, as seen by a diphenyleneiodonium-dependent suppression of basal and augmented ROS output. Moreover, this action of AGEs was found to be complementary to that of neutrophil priming agents, also known to upregulate neutrophil ROS production, implying the presence of distinct intracellular transduction pathways mediating the effect of these two classes of agents.

Advanced Glycation End Products Stimulate an Enhanced Neutrophil Respiratory Burst Mediated Through the Activation of Cytosolic Phospholipase A 2 and Generation of Arachidonic Acid

“…We previously demonstrated the ability of AGEs to augment the activated neutrophil respiratory burst. 10 Here, we investigated the effects of manipulating both AA production and intracellular redox status on this particular property of AGEs. AA is hydrolyzed from membrane glycerophospholipids by PLA 2 , 20 of which different isoforms exist.…”

“…Neutrophils were separated by dextran sedimentation and purification on a Percoll gradient. 10 Membrane and cytosol fractions were prepared according to the method of Levy and Malech. 16 Briefly, neutrophils were centrifuged down, resuspended in relaxation buffer (in mmol/L: 10 KCl, 3 NaCl, 3.5 MgCl 2 , 1.25 EGTA, 10 HEPES, pH 7.4, containing 1 mmol/L PMSF, 100 mol/L leupeptin, 10 g/mL aprotinin, 1 mmol/L Na 3 VO 4 , and 25 mmol/L NaF), snap-frozen in liquid nitrogen, defrosted, and sonicated 3 times for 15 seconds on ice.…”

“…10 Chemical stimulation was in the form of fMLP 100 nmol/L. 17 When we investigated the effects of various enzyme inhibitors on the AGE-augmented neutrophil ROS burst, neutrophils were preincubated with the respective inhibitors, after which neutrophils were spun down and resuspended in inhibitor-free buffer (containing AGE-albumin/HSA) before stimulation.…”

“…AGEs act as neutrophil coagonists, having no direct, discernible effect on neutrophil function, yet augmenting the respiratory burst induced by a secondary stimulus. 10 This is dependent on neutrophil NADPH oxidase, a multicomponent enzyme that generates superoxide. 11 The mechanism governing this AGE effect has not been characterized, although its initiation is probably receptormediated.…”

Enhanced superoxide release and elevated protein kinase C activity in neutrophils from diabetic patients: association with periodontitis

“…A well-characterized receptor for AGE is present on monocytes, macrophages, and neutrophils [15,16]. AGE alone triggers minimal O 2 − /peroxide production by neutrophils but markedly increases the production of these reactive oxygen species upon subsequent stimulation of the cells with the chemoattractant N-formyl-Met-Leu-Phe (fMLP) [16][17][18]. AGE have been implicated in susceptibility oral infections, exaggerated inflammatory responses, and destruction of alveolar bone, which accompanies diabetes [19,20].…”