Role Of Neuropeptide Y In Tumor Necrosis Factor Alpha Production Of Macrophages And The Mechanisms

Background:Neuropeptide Y(NPY),a peptide that comprises 36 amino acids,is widely distributed in the central and peripheral nervous systems,and functions as a neuromodulator and neurohormone.It plays an important role in the regulation of food intake,memory retention,cardiovascular functions and anxiety.In addition,NPY also appears to be particular important in the pathogenesis of some autoimmune diseases. NPY exerts its pleiotropic functions through the activation of several G-protein coupled NPY receptors,including Y1,Y2,Y3,Y4,Y5 and Y6 receptors.NPY and Y1 receptor are expressed by many hemopoietic and immune cells such as T cells,B cells,monocytes or macrophages.Macrophages can secrect NPY under inflammatory stimulus like LPS. Recent studies indicated that Y1^-/- macrophages produced significantly less TNF-αthan Y1^+/+ peritoneal macrophages in response to LPS;Y1R antagonists can inhibit TNF-αsecretion by normal LPS-activated peritoneal macrophages.These studies demonstrated that NPY could modulate macrophages TNF-αproduction through Y1 receptor,while the exact mechanism is still unknown.Objective:This study was to elucidate the role of NPY in macrophage proinflammatory cytokine TNF-αproduction and the possible underlying mechanisms.Methods:RT-PCR was used to characterize the mRNA expression of Y1 receptor in RAW264.7 macrophages.TNF-αin supernatants was evaluated by ELISA.Western blotting was used to detect the activity of signaling molecules.Results:NPY could promote LPS-indueed TNF-αexpression in RAW264.7 cells via Y1 receptor.RAW cells incubated with 10nM NPY showed no increased production of TNF-αversus control group;cells treated with 10nM NPY and 100ng/ml LPS showed significantly up-regulated TNF-αproduction compared with the LPS group.Addition of the Y1 receptor specific inhibitor PD 160170 before LPS and NPY prevented the up-regulation of TNF production.Stimulation of 100ng/ml LPS rapidly activated ERK1/2, JNK and p38.Addition of 10nM NPY to the cell culture medium did not affect the activation of JNK and p38,but ERK1/2 activation was enhanced;addition of the Y1 receptor inhibitor PD 160170 blocked the increased ERK1/2 activation.PI3K p85 was activated in less than 10 min,while no PKC subtype phosphorylation observed in RAW cells treated with 10nM NPY.We incubated cells with wortmannin before the addition of LPS and NPY.Both ERK activity and TNF-αproduction were detected.Incubating cells with 1μM wortmannin for 30min did not block the effect of NPY on the LPS-induced activation of ERK1/2.There was no significant difference in TNF production between the wortmannin+NPY+LPS group and the NPY+LPS group.Conclusion:Our findings indicate NPY can play a proinflammatory role in macrophages TNF-αproduction by promoting LPS-induced ERK activation via Y1 receptor.NPY could promote the ERK activation by LPS.We infer that the elevated ERK activity may partly account for the promotion of TNF production by NPY.