α7 Nicotinic acetylcholine receptor (α7 nAChR) has been found in several non-neuronal cells and is described as an important regulator of cellular function. Naturally occurring CD4+CD25+ regulatory T cells (Tregs) are essential for the active suppression of autoimmunity. The present study investigated whether naturally occurring Tregs expressed α7 nAChR and investigated the functionary role of this receptor in controlling suppressive activity of these cells. We found that CD4+CD25+ Tregs from naive C57BL/6J mice positively expressed α7 nAChR, and its activation by nicotine enhanced the suppressive capacity of Tregs. Nicotine stimulation up-regulated the expression of cytotoxic T-lymphocyte-associated antigen (CTLA)-4 and forkhead/winged helix transcription factor p3 (Foxp3) on Tregs but had no effect on the production of interleukin (IL)-10 and transforming growth factor-β1 by Tregs. In the supernatants of CD4+CD25+ Tregs/CD4+CD25− T-cell cocultures, we observed a decrease in the concentration of IL-2 in nicotine-stimulated groups, but nicotine stimulation had no effect on the ratio of IL-4/interferon (IFN)-γ, which partially represented T-cell polarization. The above-mentioned effects of nicotine were reversed by a selective α7 nAChR antagonist, α-bungarotoxin. In addition, the ratio of IL-4/IFN-γ was increased by treatment with α-bungarotoxin. We conclude that nicotine might increase Treg-mediated immune suppression of lymphocytes via α7 nAChR. The effect is related to the up-regulation of CTLA-4 as well as Foxp3 expression and decreased IL-2 secretion in CD4+CD25+ Tregs/CD4+CD25− T-cell coculture supernatants. α7 nAChR seems to be a critical regulator for immunosuppressive function of CD4+CD25+ Tregs.
Naturally occurring CD4+CD25+ regulatory T cells (Tregs) are critical for the maintenance of immunological tolerance. Their major role is to shut down T-cell-mediated immunity toward the end of an immune reaction and suppress autoreactive T cells that have escaped the process of negative selection in the thymus. For example, depletion of CD4+CD25+ Tregs leads to the spontaneous development of various autoimmune diseases in genetically susceptible animals (McHugh et al., 2001). Elimination of CD4+CD25+ Tregs enhances immune responses to non–self-antigens such as allogeneic tissue grafts (Waldmann et al., 2008) and provokes effective tumor immunity in otherwise nonresponsive animals (Shimizu et al., 1999). On the other hand, expansion of CD4+CD25+ Tregs or augmentation of their activity can suppress allograft rejection and even induce allograft tolerance (Becker et al., 2009).
Tregs have unique immunological characteristics compared with other regulatory or suppressor T cells induced by certain routes of exogenous immunization or tolerance induction. For example, they do not proliferate in response to antigenic stimulation in vitro (i.e., they are naturally anergic) (Takahashi et al., 1998), and they can potently suppress the activation and proliferation of other CD4+ or CD8+ T cells in an antigen-nonspecific manner (Thornton and Shevach, 2000). In addition, they constitutively express the α-chain of the high-affinity interleukin (IL)-2 receptor (CD25), intracellular cytotoxic T-lymphocyte antigen (CTLA)-4, and forkhead/winged helix transcription factor p3 (Foxp3) (Fontenot et al., 2003; Zhu et al., 2007). Mice lacking these key immunoregulatory molecules will exhibit lethal lymphoproliferative phenotypes (Tivol et al., 1995; Brunkow et al., 2001). Tregs are characterized by their inability to produce IL-2 and are anergic in response to antigen-specific, allogeneic, or polyclonal stimulation in vitro (Su et al., 2004). This hyporesponsiveness can be reversed in vitro by stimulation via the T-cell receptor (TCR) and high concentrations of IL-2. Indeed, IL-2 is required for expansion and suppressive function of Tregs (Setoguchi et al., 2005). Tregs suppress proliferation and cytokine production by effector T cells via a yet unidentified cell-cell contact-dependent mechanism (Dieckmann et al., 2001). Production of immunosuppressive cytokines or cell-killing molecules by Tregs, such as transforming growth factor (TGF)-β1, IL-10, granzyme B, and IL-35, might also play a role in immune depression (Vignali, 2008).
Nicotinic acetylcholine receptors (nAChRs) are composed of five receptor subunits, including α1 to α10, β1 to β4, γ, δ, and ε, which form ligand-gated ion channels (Lloyd and Williams, 2000). According to their physiological distribution, nAChRs are classified as either muscle or neuronal nAChRs (Miyazawa et al., 2003). In neurons, α7 nAChR assembles as a homopentamer composed of five individual α7 subunits that form a central pore with ligand binding at subunit junctions responsible for changes in the state of the receptor. α7 nAChR is also widely expressed in endothelial cells, enterocytes, T lymphocytes, B lymphocytes, dendritic cells, monocytes, macrophages, neutrophils, and microglia cells (Wang et al., 2003; De Rosa et al., 2005; Saeed et al., 2005). This receptor plays an important role in controlling angiogenesis, apoptosis of T cells, and the development and antibody secretion of B cells as well as down-regulating proinflammatory cytokine synthesis in macrophages (Wang et al., 2004) and glial cells. Endocytosis, cytokine production, and costimulatory molecule expression of dendritic cells are also regulated by this receptor.
Whether Tregs, an important subgroup of T lymphocytes, express α7 nAChR is still unknown. Here, we report that naturally occurring CD4+CD25+ Tregs isolated from the spleen of C57BL/6J mice express α7 nAChR and that nicotine enhances suppressive capacity of Tregs via α7 nAChR. Nicotine-induced effect might depend on up-regulation of CTLA-4 as well as Foxp3 and reduced concentration of IL-2 in the supernatant of CD4+CD25+ Treg/CD4+CD25− T-cell cocultures. These findings show that α7 nAChR is a critical regulator for immunosuppressive function of CD4+CD25+ Tregs.
Materials and Methods
Animals and Reagents.
Six-week-old male C57BL/6J mice were obtained from the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences, and Peking Union Medical College (Beijing, China). Animals were housed in separate cages in a temperature-controlled room on a 12-h light/dark cycle and allowed to acclimatize for at least 7 days before being used. All animals had free access to water but were fasted overnight before the experiment. All experimental manipulations were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, with the approval of the Scientific Investigation Board of the Chinese PLA General Hospital.
Nicotine (1-methyl-2-[3-pyridyl]pyrrolidine) and α-bungarotoxin (BGT)- fluorescein isothiocyanate (FITC) were purchased from Sigma-Aldrich (St. Louis, MO); α-bungarotoxin was purchased from Invitrogen (Carlsbad, CA). H-302, an anti-α7 nAChR antibody, was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-CTLA-4 and an isotype (1B8) (Southern Biotechnology Associates, Birmingham, AL) and anti-Foxp3 and an isotype, recombinant IgG2a (FJK-16s) (eBioscience, San Diego, CA), were used in their respective forms as FITC and PerCPCy5.5. Anti-mouse CD3e (clone 145-2C11) and anti-mouse CD28 (clone 37.51) were obtained from eBioscience. Enzyme-linked immunosorbent assay (ELISA) kits for mouse IL-10, TGF-β1, IL-2, IL-4, and interferon (IFN)-γ were obtained from Genetimes Technology Inc. (Shanghai, China).
Cells and Cell Culture.
CD4+CD25+ Tregs and CD4+CD25− T cells were isolated from murine splenocytes by magnetic cell sorting (Miltenyi Biotec, Bergisch Gladbach, Germany), according to the manufacturer's instructions. CD4+CD25+ Tregs were isolated using magnetic cell sorting in a two-step procedure. First, CD4+ T cells were pre-enriched by depletion of unwanted cells. CD25+ cells were then positively selected from the enriched CD4+ T-cell fraction. In brief, non-CD4+ T cells were depleted by indirect magnetic labeling with a cocktail of biotin-conjugated antibodies (10 μl per 107 cells, 10 min at 4°C) and then incubated with antibiotin microbeads (20 μl per 107 cells) and CD25-phycoerythrin (PE) (10 μl per 107 cells) for an additional 15 min in the dark at 4°C. The magnetically labeled cell suspension was loaded onto a column placed in the magnetic field of the magnetic cell sorter (Miltenyi Biotec). The flow-through CD4+ cells were collected and centrifuged. To isolate CD4+CD25+ Tregs, the CD25+ PE-labeled cells in the enriched CD4+ T-cell fraction were incubated with anti-PE microbeads (10 μl per 107cells) for 15 min in the dark at 4°C. The magnetically labeled CD4+CD25+ Tregs were collected after magnetic separation using two mass spectrometry columns. The purities of CD4+CD25+ Tregs and CD4+CD25− T cells were determined at 95 to 97% by FACS analysis (see Supplementary Fig. 1). Treg phenotype of these sorted cells was confirmed by Foxp3 staining of a small aliquot, and the sorted cells were found to be enriched with Foxp3+ cells. All cell counts were performed on a hemocytometer using trypan blue to exclude dead cells from the counts.
CD4+CD25+ Treg cultures were set up with 106 cells/ml in complete RPMI 1640 medium supplemented with 10% fetal calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin, and 50 μM 2-methoxyestradiol. Anti-CD3 monoclonal antibody (mAb) combined with anti-CD28 mAb at a final concentration of 1 μg/ml was added to the culture for stimulation. Nicotine, in different concentrations, was added to provide cholinergic stimulation. In some experiments, CD4+CD25+ Tregs were pretreated with α-bungarotoxin 30 min before stimulation with nicotine. At the indicated times, cell culture supernatant was collected for cytokine production assay, and the cells were used for CD4+CD25+ Treg marker analysis or suppressive activity assay.
RNA Extraction and Semiquantitative Reverse Transcription-Polymerase Chain Reaction Analysis.
Total RNA was prepared from freshly isolated CD4+CD25+ Tregs using TRIzol reagent (Invitrogen). The cDNA was prepared from 2 μg of RNA using Promega PCR Master mix (Promega, Madison, WI). Aliquots (4 μl) of cDNA were amplified by PCR using Supermix in a thermal cycler (Applied Biosystems, Foster City, CA) with specific primers. The gene-specific primer sets for α7 nAChR and β-actin were purchased from SBS Genetech (Beijing, China). The primers for mouse α7 nAChR were 5′-ATCTGGGCATTGCCAGTATC-3′ (forward) and 5′-TCCCATGAGATCCCATTCTC-3′ (reverse), generating a 199-bp α7 band. The primers for mouse β-actin were 5′-TATGCCAACACAGTGTTGTCTGG-3′ (forward) and 5′-TACTCCTGCTTGCTGATCCACAT-3′ (reverse), resulting in a PCR product of 206 bp. A sample containing all reaction reagents except cDNA was used as PCR negative control in each experiment. The absence of genomic DNA was verified using RNA that was reverse-transcribed without the enzyme (RT−). The cDNA was amplified by 40 PCR cycles (denaturation at 94°C for 3 min, annealing at 55°C for 30 s, and extension at 72°C for 1 min, with the final extension of one cycle at 72°C for 5 min). Amplified fragments of expected size were analyzed using a 2% agarose gel and were photographed under UV light.
α-BGT-FITC Binding Assays.
Freshly isolated CD4+CD25+ Tregs (2 × 105 cells) were incubated with α-BGT-FITC (0–2 μg/ml) for 15 min in FACS buffer (PBS containing 2% bovine serum albumin and 0.1% sodium azide) at 4°C in the dark. Washed cells were analyzed by flow cytometry (FACSCalibur; BD Biosciences, San Jose, CA). Data were collected and analyzed using CellQuest software (BD Biosciences). Each experiment was repeated twice, yielding similar results. Some cells were incubated with 1.5 μg/ml α-BGT-FITC in PBS for 15 min at 4°C, washed three times with PBS, and fixed for 15 min at room temperature in 4% paraformaldehyde-PBS solution, pH 7.2. After fixation, cells were washed with PBS once and mounted with antifade solution for viewing with fluorescent confocal microscopy.
Determination of α7 nAChR Protein Expression.
Cell lysates from CD4+CD25+ Tregs were analyzed for α7-subunit expression. Mouse brain tissue lysates were used as a positive control. Total protein content was estimated using the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA). Equal amounts of total protein were loaded on and separated by SDS-polyacrylamide gel electrophoresis (50 μg per lane) and then transferred to polyvinylidene difluoride membranes. Membranes were blocked with 10% defatted milk and incubated with rabbit polyclonal antibodies against the α7 subunit (1:400) overnight at 4°C, followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:2000, 1 h at 25°C), and were revealed using enhanced chemiluminescence reagents.
In Vitro Suppression.
Naive CD4+CD25+ Tregs were prestimulated with nicotine or α-bungarotoxin in different concentrations for the indicated times before use in the suppressive assay. They were mixed with naive CD4+CD25− T cells in a ratio of 1:4 (the optimal suppression ratio determined by preliminary experiments). A total of 2 × 104 cells were suspended in 200 μl of RPMI 1640 medium and stimulated with 1 μg/ml soluble anti-CD3 mAb and 1 μg/ml soluble anti-CD28 mAb in a humidified environment with 5% CO2 at 37°C. After 3 days of coculture, supernatants were collected, stored at −70°C, and analyzed by means of ELISA for T-cell cytokine production. In parallel, proliferation was measured by 3-(4,5-dimethylthythiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Experiments were performed in 96-well plates in triplicate.
Flow Cytometric Analysis.
Cultured CD4+CD25+ Tregs (106) were prepared for the flow cytometric analysis. For detection of surface CTLA-4, cells were incubated with anti-CTLA-4 antibody at a concentration of 2 μg/ml or with FITC-conjugated anti-Armenian hamster antibody as an isotype control. Staining for Foxp3 was performed according to the manufacturer's instructions. Finally, cells were fixed in 1% formaldehyde/PBS and analyzed by FACSCalibur using CellQuest software (BD Biosciences). In each case, cell fluorescence intensity obtained with a specific antibody was compared with that obtained with isotype-matched control antibodies.
Enzyme-Linked Immunosorbent Assays.
At the end of the incubation time, cell supernatants were collected, centrifuged, and stored at −70°C until tested. IL-10 and TGF-β1 levels in Treg culture supernatants were measured using ELISA kits, according to the manufacturer's instructions. To assess T-cell cytokine production, culture supernatants from CD4+CD25+ Treg/CD4+CD25− T-cell cocultures were collected, and IL-2, IL-4, and IFN-γ amounts were measured with ELISA.
Statistics were performed using SPSS software (SPSS Inc., Chicago, IL). The results were expressed as the means ± S.E.M. with the number of independent experiments run in triplicate. Analysis of variance with post hoc Bonferroni test was used in multiple comparisons. P < 0.05 was considered significant.
α7 nAChR Expression on CD4+CD25+ Tregs.
The expression of the α7 nAChR mRNA in mouse CD4+CD25+ Tregs was determined by RT-PCR. As shown in Fig. 1A, we detected a band of the expected size of 199 bp. The absence of genomic DNA contamination was demonstrated by amplifying 2 μg of total RNA from CD4+CD25+ Tregs that were reverse-transcribed without the enzyme (RT−). We analyzed the expression of α7 nAChR mRNA in mouse brain, which is known to express a high level of α7 nAChR, as a positive control. Expression of β-actin was taken as an internal control. The expression of α7 nAChR at the protein level was also established by Western blot analysis using a specific antibody for α7 nAChR, and a clear band with a molecular mass of approximately 55 kDa from either CD4+CD25+ Tregs or brain tissue was shown, with the latter used as a positive control (Fig. 1B).
To confirm further the expression of the receptor, we next stained CD4+CD25+ Tregs with FITC-labeled α-bungarotoxin, a selective α7 nAChR antagonist. FITC fluorescence was detected on the cell surface by flow cytometry and fluorescent confocal microscopy (Figs. 2 and 3).
The Effects of Nicotine on the Suppressive Activity of CD4+CD25+ Tregs.
CD4+CD25+ Tregs prestimulated with or without nicotine for 24 h were mixed with CD4+CD25− T cells at a ratio of 1:4 in the presence or absence of anti-CD3/CD28 Abs. The effects of nicotine in concentrations ranging from 0.1 to 10 μM on the proliferation of T lymphocytes are shown in Fig. 4a. CD4+CD25− T cells proliferated to a great extent when stimulated with anti-CD3 and anti-CD28 mAb. There was a decrease in proliferative capacity when cocultured with CD4+CD25+ Tregs, and the proliferation was further inhibited after nicotine stimulation at 0.1 μM. However, no change in proliferative capacity was observed in response to nicotine at the lower concentrations ranging from 0.001 to 0.01 μM (data not shown). The results suggest that 0.1 μM nicotine augmented the ability of CD4+CD25+ Tregs to suppress T cells. Increasing suppressive activity of CD4+CD25+ Tregs was more significant when prestimulated with 0.1 μM nicotine for 12 h (Fig. 4b). To confirm that the nicotine-induced enhancement of suppressive activity of CD4+CD25+ Tregs was a result of activation of α7 nAChR (using the optimal nicotine dose of 0.1 μM), CD4+CD25+ Tregs were preincubated with the selective α7 nAChR antagonist, α-bungarotoxin (0.1 μM), before nicotine stimulation. Nicotine-induced enhancement of suppressive activity of CD4+CD25+ Tregs was abrogated after α-bungarotoxin preincubation (Fig. 4c).
The Effects of Nicotine on CTLA-4 and Foxp3 Expression in CD4+CD25+ Tregs.
The effects of nicotine in concentrations ranging from 0.1 to 10 μM on the expression of CTLA-4 and Foxp3 were determined by flow cytometry after 24-h incubation of CD4+CD25+ Tregs (Fig. 5A). Nicotine increased CTLA-4 expression at 0.1 μM and had similar effects on Foxp3 expression at 0.1 and 1 μM. In addition, compared with the 24-h group, nicotine effects on CTLA-4 and Foxp3 expression were more significant in 12-h group (Fig. 5B). To determine the involvement of α7 nAChR in the nicotine actions, we examined the effects of 0.1 μM α-bungarotoxin on CTLA-4 and Foxp3 expression on CD4+CD25+ Tregs in the presence of 0.1 μM nicotine (Fig. 5, C–E). These nicotine effects were blocked by α-bungarotoxin.
Effects of Nicotine on Production of TGF-β1 and IL-10 in CD4+CD25+ Tregs.
Upon anti-CD3/CD28 Ab costimulation, CD4+CD25+ Tregs produced detectable levels of TGF-β1 but failed to produce IL-10. Nicotine had no effect on the production of TGF-β1 (Fig. 6).
Effects of Nicotine on IL-2, IL-4, and IFN-γ Production in CD4+CD25− T Cells in Coculture Experiments.
CD4+CD25+ Tregs pretreated with nicotine for 24 h were mixed with CD4+CD25− T cells. After 72 h, supernatants were harvested for analysis of cytokine production in CD4+CD25− T cells. The effects of nicotine in concentrations ranging from 0.1 to 10 μM on IL-2 production are shown in Fig. 7a. Consistent suppression of IL-2 was observed in CD4+CD25+ Treg groups. Nicotine stimulation at 0.1 μM enhanced such inhibitory response. In addition, this effect was further enhanced when pretreated with 0.1 μM nicotine for 12 h (Fig. 7b). When CD4+CD25+ Tregs were preincubated with 0.1 μM α-bungarotoxin before nicotine stimulation, the nicotine-induced effect was markedly alleviated (Fig. 7c).
We also examined the effect of nicotine on the T-cell polarization in response to CD4+CD25+ Tregs. As shown in Fig. 7, d and e, CD4+CD25+ Tregs efficiently induced type 2 T-cell polarization as demonstrated by the changes in IL-4/IFN-γ ratio, which is an indicator of Th1/Th2 balance. Nicotine stimulation had no effects on Th2-cell polarization; this was shown when it was cocultured with CD4+CD25+ Tregs. However, we found a significant reduction of IL-4/IFN-γ ratio after incubation with α-bungarotoxin (Fig. 7f). These data suggest that CD4+CD25+ Tregs might be prone to efficiently induce type 1 T-cell polarization after blocking α7 nAChR.
Here, we report that α7 nAChR was expressed on CD4+CD25+ Tregs and α7 nAChR stimulation increased CD4+CD25+ Treg-mediated suppression of the proliferation of CD4+CD25− T cells in vitro. First, the expressions of α7 nAChR mRNA and protein were shown on CD4+CD25+ Tregs using RT-PCR and Western blotting methods. We then confirmed the surface expression of α7 nAChR on CD4+CD25+ Tregs using FITC-labeled α-bungarotoxin, which is a selective α7 nAChR antagonist (Marinou and Tzartos, 2003).
Tregs play a critical role in the maintenance of self-tolerance by suppressing, in a dominant manner, immune activation of self-aggressive effector T cells. Up-regulation of Treg function or increases in the number of cells might be beneficial in the treatment of autoimmune diseases as well as allergies and for preventing allograft rejection (Wing and Sakaguchi, 2010). On the other hand, inhibiting Treg function or decreasing the number of Tregs might boost immunity against tumors and microorganisms (Belkaid, 2008; Sheu et al., 2010). In the present experiment, upon TCR stimulation, CD4+CD25+ Tregs presented in normal naive mice potently suppressed the proliferation of CD4+CD25− T cells, as reported previously, and nicotine significantly enhanced CD4+CD25+ Treg-mediated suppression of the proliferation of CD4+CD25− T cells. This finding provides a promising modality for the treatment of autoimmune disease and allergy in CD4+CD25+ Treg-targeted strategy. Nevertheless, we should note that the ultimate goal of Treg-based immunotherapy is to tip the balance between Tregs and effector T cells. How to control the nicotine effect accurately to restore the balance is the most crucial challenge. Caution should be taken; Treg therapy for bolstering self-tolerance could possibly suppress antitumor responses (Sheu et al., 2010).
Distinct mechanisms seem to mediate the suppressive effect of CD4+CD25+ Tregs. Among them, CTLA-4 signaling is crucial for CD4+CD25+ Tregs to execute their suppressive function (Wing et al., 2008; Schmidt et al., 2009). The effect of nicotine on CTLA-4 expression in CD4+CD25+ Tregs was confirmed in our study. Only CTLA-4 molecules expressed at the cell surface are able to bind molecules of the B7 family and induce signal transduction inside the T cell (Munn et al., 2004). Therefore, the ex vivo CTLA-4 surface expression that was observed implies that it plays a particularly important role for Tregs. In the current study, nicotine stimulaton up-regulated CTLA-4 surface expression on CD4+CD25+ Tregs in parallel with the up-regulation of Treg-mediated suppressive activity. Thus the up-regulation of CTLA-4 expression might result in the enhanced suppressive effect of Tregs in response to nicotine.
Foxp3 plays a key functional role in the development and functon of CD4+CD25+ Tregs, as previously shown (Fontenot et al., 2003). The absence of FOXP3 in patients with immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) or scurfy mutation results in a lack of functional Tregs (Bennett et al., 2001). Overexpression of FOXP3 in conventional T cells leads the cells to become a Treg phenotype with suppressive activity, leading to a state of immune deficiency (Aarts-Riemens et al., 2008). In fact, changes in Foxp3 expression on CD4+CD25+ Tregs were also observed in the present study. Similar to the change in CTLA-4 expression, it was noted that up-regulation of Foxp3 expression on CD4+CD25+ Tregs occurred in the presence of nicotine stimulation, suggesting that the enhanced suppressor function might be related to Foxp3 expression. In addition, the similarity of the changes in CTLA-4 and Foxp3 expression indicates a close relationship between the two markers.
Soluble factors IL-10 or TGF-β were also implicated in the suppression by Tregs in vivo, but in vitro studies unequivocally excluded the role of these two cytokines (Shevach, 2009). In this experiment, IL-10 was not detected in the culture supernatants of naturally occurring CD4+CD25+ Tregs upon TCR-mediated activation in the presence or absence of nicotine stimulation, and the result was consistent with that of Shevach (2009). Meanwhile, we found no marked changes in TGF-β production by CD4+CD25+ Tregs accompanying up-regulation of the suppressor function subsequent to nicotine stimulation. This finding therefore implies that IL-10 and TGF-β might not be involved in nicotine-induced up-regulation of Treg-mediated suppression of T lymphocytes.
Tregs could attenuate IL-2 homeostasis in two ways: inhibition of IL-2 production (Thornton and Shevach, 1998) and/or excessive IL-2 consumption (Thornton et al., 2004). Both methods are significant mechanisms of Treg-mediated suppression of CD4+ and CD8+ T cells upon TCR stimulation. With this in mind, we determined levels of IL-2 in culture supernatants from CD4+CD25+ Treg/T-cell cocultures. We show that IL-2 levels in ex vivo culture supernatants were positively correlated with the results of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium test. These results indicate that nicotine augments the ability of CD4+CD25+ Tregs to consume IL-2 and/or suppress IL-2 production, thereby leading to an enhanced CD4+CD25+ Treg-mediated suppression of CD4+ T lymphocytes.
The existence of four distinct subsets of conventional Th cells, which differ in terms of cytokine production and function, has now been firmly established: Th1, Th2, Th17, and T-follicular helper cells. Effector Th cell subsets have also been shown to suppress each other (Sun et al., 2009). In vitro evidence now suggests that Tregs suppress Th1- and Th2-type cytokine secretion (Gregori et al., 2007), and the abnormality of Tregs was related to the Th1/Th2 imbalance in injury, infection, trauma, allergy, and tumor genesis (Li and Boussiotis, 2008; Zheng et al., 2009). Our studies also assessed whether nicotine had an effect on the relationship between CD4+CD25+ Tregs and Th1/Th2 balance. In this study, CD4+CD25+ Tregs were found to efficiently induce type 2 T-cell polarization, and the result was in line with that of previous studies (Zheng et al., 2009). Stimulation of α7 nAChR on CD4+CD25+ Tregs did not markedly influence the Th1/Th2 balance, whereas type 1 T-cell polarization response to CD4+CD25+ Tregs was noted to occur after blocking α7 nAChR. We assumed that this might be due to the activation of production of unidentified cytokines by CD4+CD25+ Tregs after α7 nAChR blockage, which plays an important role in Th1-cell differentiation. Regardless of the cause, this phenomenon seems to be beneficial for the treatment of the above-mentioned diseases.
Given the complexity of the nicotinic receptors, which are formed by a great number of subunits, we focused on the potential role of the α7 receptor in regulation of suppressive activity of CD4+CD25+ Tregs. Our data reveal that nicotine enhanced the suppressive activity of CD4+CD25+ Tregs via α7 nAChR. Moreover, nicotine could inhibit the expression of CTLA-4 and Foxp3 on CD4+CD25+ Tregs through the stimulation of α7 nAChR, and the activation of α7 nAChR suppressed the concentration of IL-2 in the coculture supernatants. Taken together, these results indicate that the stimulation of α7 nAChR regulates the Treg-mediated suppression of lymphocytes.
Many selective α7 agonists have been developed and are expected to bear potential as a therapy for inflammatory diseases. These include GTS-21 [3-[(2,4-dimethoxy)benzylidene]-anabaseine], ARR17779 [(−)-spiro[1-azabicyclo [2.2.2]octane-3,5′-oxazolidin-2′-one]], and PNU-282987 [N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochloride]. Among these, GTS-21 is the most characterized (Kox et al., 2009; Rosas-Ballina et al., 2009) and, unlike nicotine, is well tolerated, with no clinically significant adverse effects in healthy male volunteers. GTS-21 may represent an important pharmacological tool for the possible translation of our findings to the clinical setting.
Because of the aforementioned findings in the present study, we mainly focused on the effects of α7 nAChR stimulation on immunosuppression of CD4+CD25+ Tregs. Future work should address the intracellular signaling pathway of α7 nAChR to identify some novel molecular targets. In addition, CD8+ T cells, dendritic cells, Th17 cells, B cells, mast cells, basophils, and eosinophils all interact with Tregs in vivo (Akdis and Akdis, 2009). The potential role and significance of α7 nAChR activation on the interaction among these cells should also be investigated to describe the overall implication of α7 nAChR stimulation on CD4+CD25+ Tregs.
In conclusion, the present study indicates that nicotine induced the expression of immunoregulatory molecules involved in the suppressive effect of T lymphocytes via α7 nAChR and weakened the survival signal of the T lymphocytes, thus leading to up-regulation of CD4+CD25+ Treg-mediated suppression. Based on the role of α7 nAChR in the suppressive activity of CD4+CD25+ Tregs, it may be feasible to use selective α7 agonists as an immunotherapy for autoimmune diseases, allergies, and allograft rejection.
This work was supported, in part, by the National Natural Science Foundation [Grants 30872683, 30971192]; the National Basic Research Program of China [Grant 2005CB522602]; and the National Natural Science Outstanding Youth Foundation of China [Grant 30125020].
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
- CD4+CD25+ regulatory T cell
- cytotoxic T-lymphocyte-associated antigen
- forkhead/winged helix transcription factor p3
- T-cell receptor
- transforming growth factor
- α7 nAChR
- α7 nicotinic acetylcholine receptor
- fluorescein isothiocyanate
- enzyme-linked immunosorbent assay
- fluorescence-activated cell sorting
- monoclonal antibody
- polymerase chain reaction
- base pair(s)
- phosphate-buffered saline
- reverse transcription-PCR
- RNA reverse-transcribed without the enzyme
- T helper
- (−)-spiro[1-azabicyclo [2.2.2]octane-3,5′-oxazolidin-2′-one]
- N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochloride.
- Received May 8, 2010.
- Accepted September 14, 2010.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics