The anti-inflammatory effects of the n-3PUFA, eicosapentaenoic acid, reveal a new and essential regulatory step in neutrophil migration across endothelial cells that requires prostaglandin-D2

The dietary n-3 PUFA, eicosapentaenoic acid (EPA) is thought to be anti-inflammatory. However, data on its ability to regulate leukocyte recruitment by vascular endothelial cells (EC) is scant. Here, primary human EC were treated with EPA for 24h prior to stimulation with TNF- and assessment of neutrophil recruitment in a flow-based assay. At levels achievable by dietary supplementation, EPA dose-dependently decreased migration of neutrophils through the EC without inhibiting their stable adhesion to the surface, a process requiring initial CXC chemokine derived signals that were unaffected by EPA. Inhibition could be replicated using inhibitors of cycloxygenase (COX-)1 and 2, while in the presence of EPA, neutrophil migration could be reconstituted by addition of arachidonic acid (AA) during the period of cytokine stimulation. These results strongly imply a role for COX generated eicosanoids in neutrophil trans-migration. Treating neutrophils with the prostaglandin-D2 [PGD2] receptor antagonist, BW868C, also dose-dependently inhibited neutrophil migration across TNF stimulated EC. Conversely, perfusion of PGD2 (derived from AA) but not PGD3 (derived from EPA) across neutrophils bound to EPA-treated EC induced them to migrate normally. Thus we describe a new PGD2-dependent step in neutrophil migration acting down stream of chemokine activation which is inhibited by EPA. Funded by the BBSRC and BHF.