Hyperglycemia-mediated PMN priming occurs through iPLA₂ activation

Introduction:

Phospholipase A₂ (PLA₂)-derived arachidonic acid activates NADPH oxidase, which catalyzes superoxide generation. Excessive superoxide release is, in part, responsible for PMN-mediated tissue damage in inflammatory diseases such as periodontitis and diabetes.

Objective:

To investigate the role of iPLA₂ in PMN-mediated superoxide release induced by hyperglycemia

Methods:

Human PMN were isolated from subjects with diabetes and healthy controls. To study the in vitro mechanism of hyperglycemia; HL-60 cells were differentiated into neutrophil-like cells with DMSO and were incubated under various hyperglycemic conditions: high glucose (HG; 25mM), S100B (RAGE ligand; 5µg/ml), or S100B+HG for a period of 24hours. Normal glucose (NG; 5.5mM) was used as control. Superoxide generation was evaluated by a cytochrome-C reduction assay with fMLP (1μM) as the stimulus. iPLA₂ expression was determined by Western blotting and quantitative real-time PCR. To study the specific role of iPLA₂ in the superoxide generation, BEL was used as a chemical inhibitor and siRNA transfection was used to knock-down iPLA₂ mRNA.

Results:

PMN from diabetic subjects showed significantly increased superoxide release compared to healthy individuals (p<0.05). Differentiated HL-60 cells cultured with S100B and S100B+HG generated significantly increased superoxide anion (2.99±0.44, 3.19±0.44 nanomoles/10^-6cells/5 min, respectively) compared to NG alone (1.76±0.37 nanomoles/10^-6cells/5 min; p<0.05). iPLA₂ expression was increased in diabetic PMN as well as cells cultured under hyperglycemic conditions compared to healthy and control cells (52% and 60%, respectively). Treatment with BEL (20µM) resulted in 58-73% reduction in superoxide generation under both normoglycemic and hyperglycemic conditions. Blocking iPLA₂ at the mRNA level with siRNA also inhibited superoxide generation in PMN.

Conclusion:

Hyperglycemia primes PMN resulting in an enhanced secretion of superoxide. iPLA₂, but not cPLA₂, derived arachidonic acid initiates activation of the oxidase suggesting that increased activity of iPLA₂ is a potential target for pharmacologic intervention. Supported by USPHS grants DE15566 and RR00533.