The n–3 polyunsaturated fatty acids contained in fish oil provide it with anti-inflammatory effects on different inflammatory diseases(Reference Kremer1, Reference Soyland, Funk and Pajka2). Several mechanisms can be involved in the influence of the degree of unsaturation of dietary fatty acids on the development of inflammatory diseases(Reference Moreno and Mitjavila3). The beneficial effects of fish oil on inflammatory diseases have been attributed to the EPA/docosahexaenoic acid (DHA) content. EPA is also substrate for AA cascade enzymes, but induced the production of alternative eicosanoids such as 3-serie prostanoids and 5-serie leukotrienes that are considered to be less pro-inflammatory compared with AA metabolites. Thus, fish oil diet reduced AA mobilization and the subsequent prostaglandin (PG)E2 synthesis(Reference Moreno, Carbonell and Sáncehz4). However, the molecular basis of beneficial effect of EPA supplementation is poorly understood as well as the comparative biological effects of AA and EPA metabolites.
Leucocyte recruitment to inflamed areas is a pivotal event in the development of the inflammatory processes(Reference Lloret and Moreno5). In this work, we studied the effects of PGE2 and PGE3 on endothelium permeability, the effects of leukotriene B4 (LTB4) and LTB5 on endothelium permeability as well as mononuclear adhesion and migration.
Endothelial monolayer permeability to albumin was measured in ECV304 cell cultures using the Casnocha et al. methodology(Reference Casnocha, Eskin and Hall6). Polymorphonuclear (PMN) granulocytes were isolated from human blood samples using Histopaque-1077. ECV304 cell confluent cultures were plated with PMN in the presence of eicosanoids and allowed to attach at 37°C for 3 h. Non-adherent cells were removed, cultures were fixed and PMN adhered to ECV304 monolayer were counted under a phase-contrast microscope. LFA-1 and MAC-1, and E-selectin and ICAM-1 expression on PMN and ECV304 surface, respectively, were analysed with a fluorescein-activated cell sorter analyser using the corresponding antibodies to each adhesion molecule. PMN chemotaxis was measured using the modified Boyden chamber technique(Reference Boyden7) and a locomotion index was calculated using Maderazo and Woronick method(Reference Maderazo and Woronick8). Results are means±se of three independent experiments performed in duplicate. Student's t test was used to determine the significance.
Our results show that both prostaglandins (PGE2 and PGE3, 0.1–100 nM) increased trans-endothelial Evans blue-albumin (EBA) permeability in a concentration-dependent manner, reaching a maximum plateau effect at 100 nM. Interestingly, the effect of PGE3 (increased 115±3% v. control) (P<0.001) was slight higher than PGE2 (90±4%) (P<0.001) action and both were significantly antagonised by EP1 (SC19200, 1 μM) and EP2 (AH6809, 1 μM) antagonist, but not by EP3 and EP4 antagonist. LTB4 and LTB5 presented a slight effect on EBA extravasation (32±2% and 21±1.5%, respectively.
LTB4 (1–100 nM) caused significant increases in the number of PMN cell adhering to endothelial cells (280±13%–405±21%) (P<0.001 in all concentrations), whereas LTB5 was not able to induce an appreciable effect. This effect of LTB4 was mediated through the enhancement of adhesion molecules such as LFA-1 and MAC-1 on PMN surface and E-selectin and ICAM-1 expression on surface of endothelial cells. Finally, we observed that LTB4 (10–100 nM) is a highly potent chemoattractant (migration index 1.25±0.05 and 1.68±0.1 v. migration index of 1 in control condition) (P<0.05 and P<0.001, respectively), whereas LTB5 presents a weak effect (migration index of 1.15±0.06 at 100 nM).
In conclusion, the summation of these differences in the LTB4/LTB5 effect on PMN transmigration may contribute to explain the beneficial impact of omega-3 in inflammatory processes.
This work was supported by the Ministerio de Ciencia y Tecnología (BFU2004-04960/BFI and BFU2007-61727/BFI) and the Generalitat de Catalunya (2005SGR0269).
