Investigation Of α7 Nicotinic Acetylcholine Receptor Expression On CD4~+CD25~+ Regulatory T Cells And The Functionary Role In Regulatory T Cell-mediated Suppression To T Lymphocytes

Objective:α7 nicotinic acetylcholine receptor (α7 nAchR) is widely expressed in lymphocytes, macrophages, microglia cells, dendritic cells (DC) and endothelial cells.α7nAchR plays an important role in controlling apoptosis of T cells, the development and antibody secretion of B cells, down-regulating pro-inflammatory cytokine synthesis in macrophages and glial cells. Phagocytosis and cytokine production of DC are also regulated by this receptor. In the present study, we investigate whether CD4+CD25+ regulatory T cells (Treg) express a7nAChR. Then stimulate or block this receptor to observe the suppressive activity of Treg and then clarify the role ofα7nAchR in Tregs mediated immune suppression to T lymphocytes.Methods:1. CD4+CD25+ regulatory T cells were isolated from C57BL/6J mouse splenocytes with a CD4+CD25+ Regulatory T Cell Isolation kit (Miltenyi Biotec). The purity of isolated Tregs was analyzed by flow cytometry.2. Expressions of a7nAChR in mouse CD4+CD25+Tregs were examined by immunofluorescence staining, Western blot, and reverse transcription-PCR, respectively.3. After being stimulated with nicotine at concentrations ranging from 0.1μM to 10μM for 24 hours, Tregs were cultured with CD4+CD25-T cells together. Proliferation of CD4+CD25-T was analyzed by MTT test. The expressions of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and forkhead/winged helix transcription factor 3 (Foxp3) molecules of Tregs were determined by means of flow cytometry. IL-10 and TGF-β1 secretion collected in the Tregs suspension were determined by means of ELISA. By collecting the suspension of Tregs/CD4+CD25-T cell cocultures, the different kinds of cytokines were examined by means of ELISA, including IL-2, IL-4 and interferon (IFN)-y. Finally, the dose-dependent responses between nicotine and suppressive activity of Tregs were defined.4. After being stimulated with nicotine for different times ranging from 12h to 36h, the time-dependent responses between nicotine and suppressive activity of Tregs were defined by detecting the above mentioned indicators using the same methods.5. Tregs were treated with a selective a7nAChR antagonist, a-bungarotoxin(a-Bgt), in the presence of nicotine. Whether a-bungarotoxin can reverse nicotine-induced effects was observed.Results:1. The purity of CD4+CD25+Tregs was above 90% after isolated twice by magnetic beads, and the activity of Treg exceeded 97%.2. It was revealed by flow cytometry that Tregs could bind a-Bgt-FITC. Moreover, a positive binding to a-Bgt was also observed on the cell surface of Treg, as viewed by fluorescent confocal microscopy. In addition, a clear band ofα7nAChR with a molecular mass of approximately 55 kD was found from Tregs by Western blot analysis, andα7nAChR mRNA was expressed with the expected size of 199-bp from Tregs by reverse transcription-PCR.3. The suppressive activity of Tregs was markedly increased when stimulated with nicotine at 0.1μM (P<0.01). The expression of CTLA-4 was significantly enhanced when Tregs were stimulatd nicotine at 0.1μM (P<0.01). The expression of Foxp3 was significantly enhanced when Tregs were stimulatd nicotine at 0.1μM or 1μM (P<0.01). After stimulation with nicotine at concentrations ranging from 0.1μM to 10μM, IL-10 and TGF-β1 secretion were unchanged. After being stimulated with nicotine at 0.1μM, concentrations of IL-2 in the suspension of Tregs/CD4+CD25-T cell co-cultures were decreased(P<0.05). Moreover, the ratios of IL-4/IFN-y concentration in the suspension were unchanged when stimulated with nicotine at concentrations ranging from 0.1μM to 10μM.4. Compared with unstimulated-Treg group, the suppressive activity of proliferation of CD4+CD25-T cells was increased when stimulated with nicotine at 0.1μM for 12h or 24 h(P<0.01 or P<0.05). Further investigation revealed that both CTLA-4 and Foxp3 expressions were significantly up-regulated at 12 h or 24 h (P<0.01 or P<0.05). After stimulation with nicotine for 12 h to 36 h, IL-10 and TGF-β1 secretion were unchanged. In the suspension of Tregs/CD4+CD25-T cell co-cultures in 12 hour groups, the concentrations of IL-2 markedly decreased (P<0.05) and the ratios of IL-4/IFN-y concentration was not markedly changed.5. The effects of nicotine upregulating suppressive activity of Tregs was reversed by a-Bgt (P<0.01). The effects of nicotine upregulating CTLA-4 and Foxp3 expressions was also reversed by a-Bgt (P<0.01). When CD4+CD25-T cells were cultured with stimulated Tregs, in comparsion with unstimulated-Treg group, the levels of IL-2 was decreased. When co-stimulates Tregs with nicotine and a-Bgt, concentrations of IL-2 recovered to the level of un-stimulated groups. Meanwhile the ratios of IL-4/IFN-y concentration were significantly decreased when cultured with stimulated Tregs by a-Bgt (P<0.01).Conclusion:1. Natural CD4+CD25+ Tregs from mice express a7nAChR.2. Interacting with a7nAChR, nicotine can upregulate expressions of suppressive molecules CTLA-4 and Foxp3, decrease the concentrations of IL-2 in the suspension of Tregs/CD4+CD25-T cell co-cultures and then increase the suppressive activity of CD4+CD25+ Treg cells.3. Selective a7nAChR antagonist could reverse nicotine-induced effects. Thus,α7nAChR is involved in the suppressive activity of Tregs.4. Tregs induced type 1 T cell-polarization after a7nAChR was blocked.