Investigation On JAK3-STAT5 Signaling Pathway Downstream Of The α7 Nicotinic Acetylcholine Receptor Located Of The Surface Of The Mouse CD4~+CD25~+ Regulatory T Cells

Objectives:Lymphocytes have their own independent cholinergic system, which includes acetylcholine (ACh), choline acetylase (ChAT), acetylcholinesterase (AChE), muscarinic ACh receptor (mAChR) and nicotinic ACh receptor (nAChR). The a7 nicotinic acetylcholine receptor (a7nAChR) that plays an important role in the cholinergic anti-inflammtory pathway is expressed by the immature T lymphocytes in mouse thymus gland, CD3 positive T cell in rat spleen and multiple T cell strains. Former study has confirmed that mouse CD4+CD25+ regulatory T cell (Treg) expressed a7nAChR on its surface, and activation of this receptor could enhance the suppressive function of Treg on other kinds of immune cells. The present study investigated the association between a7nAChR and the JAK3-STAT5 signaling pathway in mouse CD4+CD25+ Treg.Methods:1. CD4+CD25+ regulatory T cells from the spleen of C57BL/6J mice were separated by means of MACS microbeads and then identified for purity and activity using flow cytometry and trypan blue staining; 2. Pretreating these cells with nicotine (agonist of a7nAChR) and a-bungarotoxin (receptor's specific antagonist), then we co-cultured them with CD4+CD25- T cells whose proliferative situation was then assessed by MTT assay; 3. Treg cells were tested for the expression of signaling pathway protein JAK3 and STAT5 through confocal microscopy and western blotting; 4. Similarly pretreating CD4+CD25+ Treg cells with nicotine and a-bungarotoxin, we evaluated the expression changes of JAK3 and STAT5 protein with western blot test.Results:1. The purity of the separated CD4+CD25+ Treg cells was above 90% and more than 97% of which were alive; 2. MTT assay showed that Treg cells which were treated with 10μmol/L nicotine had higher suppressive effect on CD4+CD25- T cells' proliferation compared with control group (untreated Treg cells), and Tregs which were treated with 1μmol/L a-bungarotoxin alone or with nicotine and a-bungarotoxin co-administration had no such effect; 3. Confocal microscopy assay showed that there were JAK3, STAT5, p-JAK3 and p-STAT5 proteins expressed in the cytoplasm of CD4+CD25+ Treg. Furthermore, Western blot test got protein strips of JAK3, STAT5, p-JAK3 and p-STAT5 whose molecular weight were 106kD,106kD,92kD and 94kD respectively in Treg cell's gross protein extracts; 4. Pretreated with nicotine, CD4+CD25+ Treg cells expressed more p-JAK3 and p-STAT5 proteins than untreated ones, but a-bungarotoxin alone or nicotine and a-bungarotoxin co-administration treatment had no such effect mentioned above.Conclusions:1. The results suggest that activation of a7nAChR may enhance the immunosuppressive function of CD4+CD25+ Treg cells; 2. It is found that JAK3, p-JAK3, STAT5 and p-STAT5 proteins be expressed in CD4+CD25+ Treg cells.3. Nicotine may enhance the expression of p- JAK3 and p-STAT5 in Treg cells, however, such effect can be reversed by the specific antagonist of a7nAChR a-bungarotoxin.