NE Increases Inflammatory Factors Production By Macrophage Through Modulating TLR4

It is generally accepted that stress can affect the immunity of the body notably. More and more researchers have paid so much attention to this. But most of the former research focused on the glucocorticoid (GC) what came from the adrenal cortex. On the other hand, less research work involved another important stress-related hormone NE. It was widely recognized that NE could aggravate many kinds of diseases such as LPS shock, allergic disease, inflammatory disease and tumor and so, yet the mechanism remained absolutely unclear. It would be very instructive and helpful for the human being to study this concretely.It was well known that NE in the circulation of the body mostly came from the sympathetic nerve terminal. Especially when the body was under stress state, the brain excited the sympathetic nerve and a great deal of NE could be released into the circulation. Terminal of sympathetic nerve almost spread all over of the body. Besides the cardiovascular system and nervous system, sympathetic nerve could be found everywhere in each immunity organ. Through releasing NE into the immunity organs, the sympathetic nerve system manipulated the immunity system profoundly. What's more, between the immunity organs and the sympathetic terminal, there existed an interesting special structure which resembled the"synapse". Macrophage could be found in each immunity organ, and the "synapse" was so close to it definitely. Nevertheless, almost all immune cells expressed adrenergic acceptors. All of these conditions suggested that the sympathetic nerve system and immune system related to each other closely, and it would be very easy to be affected by each other. All of these structures built up a material foundation for the sympathetic nerve system to regulate the function of macrophage. And there was still another interesting thing that when macrophage was treated by LPS, macrophage could release so much NPY and NE and so on. Then when being under stress state or some kind of infection situation, macrophage could be so easily flooded and influenced by NE. But how NE precisely took effect on macrophage still remained unknown, especially when the body was under stress state.Macrophage exerted so many complicated functions in the body though several different ways. It could affect almost all of the diseases such as inflammatory diseases and tumors and so on. There were many toll-like receptors 4 on the membrane of the macrophage, which were the natural receptor of LPS. TLR4 was the key point of the function of macrophage. When TLR4 was stimulated, a great deal of cytokines would be produced and released out of macrophage, such as TNFα, IL-1, IL-6 and IFN and so on. Most of these had important functions on the immunity and inflammation reaction. What's more, many factors could regulate TLR4 such as MIF, hypooxygen, and O³ and so on. Among these, ROS was the key point of modulating TLR4. By the way, NE could lead to oxidative stress in cardiovascular system evidently. Then maybe NE could affect the oxidative state of the macrophage? And then NE could modulate TLR4 though ROS?Above all, till now there are so many unknown areas about how sympathetic nerve system regulates the function of the immunity, and through which key point it exerts its action. In one word, we still have so long a way to go. What does NE which came from the sympathetic terminal mean to macrophage? And, how does NE take effect on macrophage? Maybe NE implements its function through modulating TLR4 by ROS? In order to prove this hypothesis, we designed a serial of experiments to test it.(1)We applied the immunohistochemistry technique to investigate whether NE could modulate the expression of TLR4 in the spleen after the mice subjected to the restrained stress process. And we also determined the survival rate in 72 hours of the stressed mice when they experienced LPS shock.(2)We applied Western blot and Quantitive real time PCR to test if NE could affect the expression of TLR4 in vitro.(3)We applied Western blot to test whether ROS could take effect on the expression of TLR4 in vitro, and then applied cytochrome C revivification method and fluoresce probe DCFH-DA to test if NE could affect the redox state of macrophage in vitro.(4)After being pretreated with NE or ROS, we applied ELISA and chemiluminescence method to determine the production of TNFa and NO after the macrophage was stimulated by LPS.(5)At last we explored if NE could stimulate the phosphorylation of the PKC of macrophage.The results were listed as follows:(1) NE could affect the expression of TLR4. The immnohistochemistry results showed us that the stressed animal spleens presented much more TLR4 expression than control and 6-OHDA+stess group. The survival rate of stressed animals which were being pretreated with 6-OHDA was higher than that in stressed animals.(2) NE could promote the expression of TLR4. Andβadrenoreceptor inhibitor propranolol, a adrenoreceptor inhibitor phentolamine and N-Acetylcysteine all could partly antagonize NE's effect.(3) NE could regulate the ROS production of macrophage. Through cytochrome C revivification method and fluorescence probes method, the results showed that NE could stimulate macrophage to produce much ROS. And ROS could modulate the expression of TLR4 in macrophage bidirectionally. An appropriate dose of H₂O₂ could increase the expression of TLR4, but higher level of H₂O₂ could decrease the expression of TLR4, yet much lower level had no effect on it.(4) The result of ELISA and chemiluminescence showed us that macrophage pretreated by NE and H₂O₂ produced more TNFαand NO than that in control group.(5) NE could stimulate the phosphorilation of PKC. Theβadrenoreceptor inhibitor propranolol andαadrenoreceptor inhibitor phentolamine could partly antagonize its effect.