Resolvin E1 on PKC Activity in Neutrophils from LAP Patients

Objective: Protein Kinase C (PKC), a critical second messenger in cellular signaling has been implicated in the assembly of NADPH oxidase and the generation of superoxide in neutrophils (PMN). In Localized Aggressive Periodontitis (LAP), PMN generate excess superoxide leading to tissue destruction. Resolvin E1 (RvE1), an inflammation resolving lipid agonist, has been found to reduce superoxide generation and play a key role in the resolution of both acute and chronic inflammation. The aim of this study is to examine the kinetics of intracellular PKC and p47phox activation in LAP and healthy PMN treated with RvE1 and to determine phosphorylation of PKC isoforms and p47phox.

Methods: Neutrophils from LAP and matched healthy subjects were isolated by Ficoll-Hypaque density gradient centrifugation, incubated with RvE1 (1 nM) or PBS, and stimulated with fMLP (1 µM) for 0, 30, and 60 seconds. Samples were analyzed by Western blotting with antibodies against phospho-PKC a, b1, b2, d, and z and phospho-PKC(S). Superoxide generation was measured using a cytochrome c reduction assay.

Results: LAP neutrophils secrete elevated superoxide anion. RvE1 inhibited the phosphorylation of PKC b2, d, z and p47phox in LAP neutrophils by a fold of 0.75, 0.73, 0.51, and 0.53 from the baseline, respectively. RvE1 exhibited no action in normal neutrophils. RvE1 treatment of LAP neutrophils reversed the primed phenotype and returned superoxide generation levels to normal.

Conclusion: Pre-activation of PKC isoforms in LAP plays an important role in priming of PMN. LAP PMN are primed in situ as evidenced by phosphorylation of the major PKC isoforms. RvE1 suppressed the phosphorylation of PKC b2, d, and z and p47phox in PMN from LAP and reversed abnormal superoxide generation suggesting that RvE1 and related molecules have potential in the pharmacologic management of inflammatory diseases such as periodontitis.

Supported by USPHS Grants DE016191 and RR00533.