Effects Of Lymphocytes-Derived Catecholamines On The Differentiation Of T Helper Cells

Objectives:With the increasing studies on the neuro-endocrine immune interaction, we found that rat and human immune cells can both express tyrosine hydroxylase (TH), a rate-limiting enzyme of catecholamines(CAs) synthesis. Subsequent studies have also confirmed the lymphocytes themselves can secrete CAs and have an effect on their own proliferation and apoptosis. And on this basis, researchers have done a preliminary exploration on mechanisms of the action. T lymphocyte is a highly heterogeneous group, among which CD4+T cells are in the majority. The aim of this study is to explore the role of the CAs secreted by the Th cells themselves on their own differentiation so as to extend the comprehension of neuro-endocrine-immune regulatory network.Methods:CD4+T lymphocytes were separated from mesenteric lymph nodes of mice and purified with Magnetic bead method. Reverse transcription-polymerase chain reaction (RT-PCR) was used to measure (?) the mRNA expression of TH in purified CD4+T lymphocytes and western blot was employed to measure the protein expression of TH in CD4+T lymphocytes. Four pairs of pcDNATM6.2-GW/miRNA corresponding to TH (NM₀09377.1) were constructed and transfected into CD4+T lymphocytes using AMAXA neucleofector technology. Silencing effects of TH were detected by real-time PCR and western blot methods. The intracellular CAs of the CD4+T lymphocytes were detected by high performance liquid chromatography with electrochemical detection (HPLC-ED) after TH was down-regulated. To assess intracellular cytokines interferon-y (IFN-y) and interleukin-4 (IL-4), flow cytometry method of intracellular cytokine staining was performed after TH was silenced down.Results:After the lymphocytes were separated and purified from mesenteric lymph nodes of mice, we found that the cells could express TH mRNA and protein. Sequence of expression vector pcDNATM6.2-GW /EmGFP-miR-TH was proved correct using sequencing method. The transfect rate of pcDNATM6.2-GW/EmGFP-miR-TH into CD4+T lymphocytes using AMAXA neucleofector technology could reach 60%-70%. After the vector pcDNATM6.2-GW/EmGFP-miR-TH being transfected into CD4+T lymphocytes, we observed TH mRNA and protein level was down regulated by 60-80%, of which the miRNA(4) sequence had the best silencing effect. The result of HPLC-ED showed the contents of intracellular CAs were decreased siginificantly after TH was silenced down.The results of flow cytometry method of intracellular cytokine staining showed that a significant decrease in the percentage of IL-4-producing T cells and no evident change in the percentage of IFN-y-producing T cells was found in the group of TH silenced down, resulting in a higher ratio of IFN-y/IL-4 producing T cells compared with control.Conclusions:CD4+T lymphocytes can express TH. Down-regulation of TH in CD4+T lymphocytes can suppress the content of intracellular CAs. Th-derived CAs promote the polarization of immune response towards the Th2 type.