Study On Effects Of Melatonin And Its Membrane Receptor On CD4+T Cells Activation In Adults

As a hormone of pineal, melatonin (MLT) not only involved in the regulation of circadian rhythm, at the same time also acted on immune system, immune organs, immune cells, and immune molecules, thereby affected the regulation of immune function. After secreted into the blood, MLT started up biological effects mainly binding with MLT receptor (melatonin receptor MR). In 1998 IU PHAR meeting, people named human and other mammals'MLT receptor as MT₁ (melatonin receptor, new name), MT₂ and MT₃. MT₁ and MT₂, respectively, corresponding to the previous Mel la (melatonin receptor, old name) and Mel lb, MT₃ is equivalent to the past Mel 2.CD (cluster of differentiation) generally refers to a class of differentiation antigen clusters on the surface of leukocyte. As the important adherent, discriminating components of the lymphocyte surface, plays an important role in immune regulation. CD69 is known as an activation-induced molecules (activation inducer molecule, A IM), or the early activated antigen-l (EA-1), is a member of C-type lectin receptor families, CD69 is early activation markers. CD25 is a T lymphocyte surface marker relatively early, is one kind of antigen on the surface of the activated T lymphocytes. HLA-DR is a MHC-â…¡molecules, one of the major antigen, HLA-DR has a low level expression in resting T-cells. It increases slowly and lower by stimulation, so it is the later activation antigen on T cell, but expression is constant. Therefore, as the activated molecular markers of T cell, CD25, CD69 and HLA-DR can be used to assess the T cell's activation.CD4+ T cell is a class of helper T lymphocyte (T helper cell, Th cell), has an important immune regulatory function, and its activation plays a key role in the immune process, so it has great significance for the diagnosis and treatment of many immunologic diseases. Melatonin can affect the Th cells transformation, promote or inhibit the secretion of cytokines, which have a lot of coverage. However, how melatonin effects on the process of T cell activation,which should to be clear.Objective: To investigate the role of melatonin and its membrane receptor effecting the activating process of CD4 + T cell in adults.Methods:1. The effects of melatonin on CD4 + T cell activation and the establishment of its optimal concentration10 cases of isolated peripheral blood lymphocytes from random selected normal and healthy adults, each specimen was divided into 4 groups with the different concentration of melatonin, added the MLT, and the concentrations were separate 0 pM, 100 pM, 500 pM and 1 nM. With the inhibition of melatonin synthesis by the parachlorophenylalanin (PCPA, 10μM) in lymphocytes and PHA's stimulation(8μg/ml), these peripheral blood lymphocytes were cultured. After 20 hours, take some cultured cells to detect the CD4+CD69+ T cell `s percentage of the total CD4+ T lymphocytes by flow cytometry, 72 hours later analysised the remainder of the cultured cells to know the CD4+CD25+ and CD4+HLA-DA+ T cell percentage of the total CD4+ T lymphocytes. By paired t-test, to know whether melatonin has the effects on CD4+ T cell, and whether there is obvious dose-effect relationship, then to determine the best concentration of MLT.2. The role of melatonin and its receptor antagonists on the activation of CD4 + T cellRandom selected 27 cases of isolated peripheral blood lymphocytes from normal adults, each specimen divided into 5 groups with the different Processing factors, first group is the control, the second group added MLT(1 nM), the third group was 2-phenyl-N-acetyl serotonin (lzindole, LZI, MT1/MT₂ non-selective inhibitor, 10μM)and MLT(1 nM), the fourth group was Prazosin hydrochloride(PRZ, MT₃ inhibitor, 20.4 nM) and MLT(1 nM), the fifth group mixed in LZI(10μM), PRZ(20.4 nM) and MLT(1 nM). With the inhibition of melatonin synthesis by the PCPA (10μM) in lymphocytes and PHA's stimulation, these peripheral blood lymphocytes were cultured. After 20 hours, take some cultured cells to detected CD4+CD69+ T cell percentage the total CD4+ T lymphocytes and mean fluorescence index(MFI) by flow cytometry, 72 hours later analysised the remainder of the cells by flow cytometry to know the CD4+CD25+ and CD4+HLA-DA+ T cell's percentage of the total CD4+ T lymphocytes and the MFI. By ANOVA test, observe the role of melatonin and its receptor antagonists on the .activation of CD4+ T cell.Results:1. The inhibition of melatonin synthesis by the parachlorophenylalanine(PCPA) and the lymphocytes were stimulated by PHA, the exogenous melatonin can effectively stimulate CD4+ T cells and increase the percentages of CD4+CD25+, CD4+CD69+, CD4+HLA-DR+ T cells (P <0.05), and the percentages of these cells increased with the increasing concentrations of melatonin(P<0.05);2. But the MFI of CD4 + CD25 + T cells, CD4 + CD69 + T cells and CD4 + HLA-DR + T cells have no significant difference(P>0.05).3. Lzindole can effectively suppress the above-mentioned role of melatonin (P <0.05), but prazosin hydrochloride can not inhibit such role.Conclusion:1. These findings suggest that the exogenous melatonin can effectively enhance the CD4+ T cells'activation by PHA in adults, and obviously there is a dose-effect relationship;2. MLT cannot raise the amount of CD25, CD69, HLA-DR molecule expressing on the activated CD4+ T cell.3. Melatonin plays the role through MT₁/MT₂, but not MT₃.