GPR40 EXPRESSION AND FUNCTION IN IMMUNE CELLS AND EXPERIMENTAL ARTHRITIS
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Omega-3 fatty acids (ω-3 FA, including eicosapentaenoic acid [EPA] and docosahexaenoic acid [DHA]), are essential polyunsaturated fatty acids which are correlated with lower incidence of chronic diseases. DHA and EPA can be enzymatically converted to resolvins, protectins and maresins, which play important roles in resolution of inflammation. Additionally, ω-3 FA can also directly activate surface receptors, namely the long-chain free fatty acid receptors GPR40 and GPR120, two GPCRs with poorly investigated biology. Using real-time PCR analysis, GPR40 transcript in human neutrophils was detected; the protein expression was also confirmed by flow cytometry and image stream analysis. Expression of GPR40 protein was up-regulated after stimulation with platelet-activating factor (PAF, 10nM) or leukotriene B4 (LTB4, 10nM) for 10 minutes. I utilised the selective agonist GW9508 to investigate the biology of GPR40. Tested on human neutrophils, GW9508 elevated intracellular calcium when applied within the 0.1-10μM range. The up-regulation of GPR40 expression by pro-inflammatory stimuli suggested to us potential regulatory roles for this receptor during inflammation. I then showed that 1 and 10μM GW9508 increased neutrophil chemotaxis in response to the cytokine IL-8 (30ng/ml). In addition, GPR40 activation by GW9508 enhanced phagocytosis of E. coli by human neutrophils by approximately 50% when tested at 0.1 and 1μM. Moreover, GW9508-neutrophil stimulation augmented microvesicle release and delayed apoptosis after stimulation. Finally, I demonstrated that GPR40 is expressed in inflammatory cells isolated from murine arthritic joints, such as neutrophils, macrophages and inflammatory monocytes. KBN-serum induced arthritic mice developed a more severe disease when treated prophylactically with GW9508 (10mg/kg, i.p. treated from day 0, daily), characterized by a higher clinical score and increased oedema when compared to vehicle control mice. Therapeutic intervention with GW9508 at the peak of the disease (day 5) delayed the resolution of arthritis. In summary, the data suggest that activation of GPR40 by GW9508 enhances neutrophil activation, up regulating the pro-inflammatory properties of this cell type, and therefore, exacerbating experimental inflammatory arthritis.
Authorsde Souza, Patricia Regina Soares
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