Expression and Functional Role of α7 Nicotinic Receptor in Human Cytokine-stimulated Natural Killer (NK) Cells*

The homomeric α7 nicotinic receptor (nAChR) is one of the most abundant nAChRs in the central nervous system where it contributes to cognition, attention, and working memory. α7 nAChR is also present in lymphocytes, dendritic cells (DCs), and macrophages and it is emerging as an important drug target for intervention in inflammation and sepsis. Natural killer (NK) cells display cytotoxic activity against susceptible target cells and modulate innate and adaptive immune responses through their interaction with DCs. We here show that human NK cells also express α7 nAChR. α7 nAChR mRNA is detected by RT-PCR and cell surface expression of α7 nAChR is detected by confocal microscopy and flow cytometry using α-bungarotoxin, a specific antagonist. Both mRNA and protein levels increase during NK stimulation with cytokines (IL-12, IL-18, and IL-15). Exposure of cytokine-stimulated NK cells to PNU-282987, a specific α7 nAChR agonist, increases intracellular calcium concentration ([Ca2+]i) mainly released from intracellular stores, indicating that α7 nAChR is functional. Moreover, its activation by PNU-282987 plus a specific positive allosteric modulator greatly enhances the Ca2+ responses in NK cells. Stimulation of NK cells with cytokines and PNU-282987 decreases NF-κB levels and nuclear mobilization, down-regulates NKG2D receptors, and decreases NKG2D-dependent cell-mediated cytotoxicity and IFN-γ production. Also, such NK cells are less efficient to trigger DC maturation. Thus, our results demonstrate the anti-inflammatory role of α7 nAChR in NK cells and suggest that modulation of its activity in these cells may constitute a novel target for regulation of the immune response.

vance of ␣7 nAChR as a novel immunomodulatory target, its presence in human NK cells has not been yet determined.
NK cells constitute 5-15% of lymphoid cells in human peripheral blood and are critical during immunity against intracellular infectious agents and tumors (18,19). NK cells have also immunoregulatory properties due to the secretion of multiple cytokines such as IFN-␥, TNF-␣, GM-CSF, and chemokines that contribute to modulate the innate immune response and to shift the adaptive immune response to a Th1-biased profile through interactions with DCs and macrophages (18, 20 -23). Accumulating evidence indicates that NK cells may also play an important role during autoimmune disorders (19,24,25). The activity of NK cells is controlled by a dynamic balance of signals elicited upon engagement of activating and inhibitory receptors by discrete ligands expressed on target cells (23,26). Very limited information suggests that cholinergic receptors may modulate NK cell activity (27,28). It has been demonstrated that mouse NK cells express ␤2 nAChR subunit and that this subunit is involved in nicotine-induced attenuation of mouse NK cell functions (29).
Therefore, the aim of this work was to investigate the expression of ␣7 nAChR in human NK cells and the regulation of NK cell effector functions by ␣7 nAChR ligands. We report here the first experimental evidence demonstrating the expression of ␣7 nAChR on human NK cells and the biological consequences of its activation.

Results
Human NK Cells Express ␣7 nAChR-First, we isolated NK cells from healthy donors and assessed the expression of ␣7 nAChR mRNA by RT-PCR. As shown in Fig. 1A, freshly isolated human NK cells express ␣7 nAChR mRNA. By analogy with other immune cells, we hypothesized that the expression of nAChRs in NK cells changes upon cell activation (8, 30 -32). As human NK cells become activated by cytokines, particularly IL-12, IL-18, and IL-15 (23,33,34), and considering that these cytokines increase during inflammation, we stimulated NK cells with these cytokines and performed a kinetic analysis of ␣7 nAChR mRNA expression by quantitative PCR (qPCR). The results show that ␣7 nAChR mRNA peaks at 48 h after cell stimulation and returns to background levels after 72 h (data not shown). The peak corresponds to a ϳ3.1-fold increase in ␣7 nAChR mRNA with respect to that of fresh cells (Fig. 1B). Therefore, we chose 48 h stimulation with cytokines for all our experiments.
We also detected in freshly isolated NK cells mRNA corresponding to ␣4 (Fig. 1C) and ␤2 (Fig. 1E) nAChR subunits. By qPCR we determined that after cytokine stimulation of NK cells, the mRNA level of ␣4 nAChR does not change significantly (Fig. 1D), whereas that of ␤2 slightly increases with respect to fresh cells (Fig. 1F). As different nAChR types are present in human NK cells, we used a selective ␣7 nAChR agonist (PNU-282987) and a specific antagonist (␣-BTX) for further functional experiments.
Cell surface expression of ␣7 nAChR was analyzed using Alexa Fluor 488-labeled ␣-BTX, a selective ␣7 nAChR antagonist, in NK cells cultured in the absence or presence of IL-12, IL-18, and IL-15 for 48 h and assessed by fluorescence micros-copy ( Fig. 2A) and flow cytometry (FC) (Fig. 2B). In line with the qPCR results, an increased number of fluorescent cells and higher fluorescence per cell are observed on stimulated NK cells compared with resting cells. The pretreatment with ␣-BTX markedly reduces the binding of Alexa Fluor 488-labeled ␣-BTX to the cells (Fig. 2). These results indicate that NK cells express surface ␣7 nAChR that is up-regulated during NK stimulation with cytokines.
Activation of ␣7 nAChR Triggers Ca 2ϩ Mobilization in Human NK Cells-To investigate whether ␣7 nAChR in NK cells is functional and its activation triggers the typical changes in [Ca 2ϩ ] i , we conducted confocal microscopy analysis of NK cells cultured in the presence of cytokines during 48 h and loaded with Fluo-3/AM. PNU-282987 leads to a slow and continuous increase of [Ca 2ϩ ] i (Fig. 3, B, E, and F). Although the magnitude of the responses vary among individual cells, all cells show a clear change in [Ca 2ϩ ] i after agonist addition (Fig. 3, A  and B). The Ca 2ϩ responses are not detected in resting cells, probably due to the lower expression level of ␣7 nAChR as also described in lymphocytes (8,31).
To further confirm that the increase in [Ca 2ϩ ] i is mediated by ␣7 nAChR, we co-applied PNU-282987 with PNU-120596, which is a specific ␣7 nAChR positive allosteric modulator (PAM) (35). As expected, the presence of the PAM leads to an enhanced increase of [Ca 2ϩ ] i (Fig. 3, C and G), which is revealed by a slight increase in maximum fluorescence intensity (Fig. 3I) FIGURE 1. Human NK cells express mRNA for the ␣7, ␣4, and ␤2 nAChR subunits. Expression of ␣7, ␣4, and ␤2 nAChR subunits mRNA was assessed in human NK cells by RT-PCR. A, C, and E, transcripts of ␣7, ␣4, and ␤2 nAChR subunits were detected by electrophoresis in agarose gel as bands of 75, 121, and 136 bp, respectively (arrows); M, DNA ladder (50 bp (A) and 100 bp (C and E)); 1, PCR negative control; 2, PCR positive control (A, human ␣7 cDNA; B, human ␣4 cDNA; C, human ␤2 cDNA); 3, human NK cells. B, D, and F, relative quantification (RQ) of mRNA expression of ␣7, ␣4, and ␤2 nAChR subunit mRNA in human NK cells incubated with and without IL-12, IL-18, and IL-15 for 48 h. qPCR was carried out as described under "Experimental Procedures." The results are the mean Ϯ S.E., and are cumulative of at least three independent experiments performed with NK cells from different donors (n ϭ 3-5). *, p Ͻ 0.05; ***, p Ͻ 0.001; paired t test.
and an increase in the rate of change in Ca 2ϩ signal (Fig. 3, F and G). The area under the curve is 20% bigger in the presence of the agonist and PAM with respect to the agonist alone. Moreover, the change in [Ca 2ϩ ] i induced by PNU-282987 is abolished if cells are preincubated 15 min with 1 M ␣-BTX, again confirming that the effect is specifically mediated by ␣7 nAChR activation (Fig. 3, D, H, and I).
To further elucidate the origin of the [Ca 2ϩ ] i increased by PNU-282987, we stimulated human NK cells with this agonist in Ca 2ϩ -free buffer containing 5 mM EGTA, an extracellular Ca 2ϩ chelator. The PNU282987-elicited increase in [Ca 2ϩ ] i is not affected by the absence of extracellular Ca 2ϩ (Fig. 4, A, C, and E). In contrast, the increase in [Ca 2ϩ ] i is inhibited by preincubation of cells with 5 M BAPTA-AM, a cell-permeable intracellular Ca 2ϩ chelator (Fig. 4, B and D). These results demonstrate that ␣7 nAChR in NK cells is functional and its activation releases Ca 2ϩ from intracellular stores, which, in turn, may modulate cell function.
Activation of ␣7 nAChR in Human NK Cells Regulates NKG2D Expression-We next examined whether activation of ␣7 nAChR affects the expression of some major NK cell activating receptors such as NKG2D, NKp46, and DNAM-1. To achieve maximal effects, we first incubated NK cells with IL-12, IL-18, and IL-15 for 48 h to promote maximal ␣7 nAChR expression. Thereafter, we treated the cells for 18 h in the presence of the cytokines with the agonist and/or antagonist, and assessed their effects on the expression of the NK cell receptors by FC. We observed that PNU-282987 induces down-regulation of NKG2D, and is abolished by preincubation of cells with ␣-BTX (Fig. 5A). In contrast, the expression levels of the other NK cell receptors, NKp46 and DNAM-1, are not affected by the presence of PNU-282987 (Fig. 5, B and C). Of note, PNU-282987 does not affect NK cell viability (11.3 Ϯ 2.2% of cell death in control versus 12.9 Ϯ 2.1% in PNU-282987-treated cells). These experiments indicate that activation of ␣7 nAChR down-regulates selectively the expression of NKG2D, which is one of the major activating receptors involved in NK cell function.
␣7 nAChR Activation Affects NK Cell-mediated Cytotoxicity, IFN-␥ Production, and Impacts on Maturation of Human DCs-To determine the physiological consequences of activation of ␣7 nAChR on NK cells, we assessed their NKG2Ddependent cytotoxic activity and IFN-␥ production. In both studies, NK cells were stimulated with cytokines for 48 h and further cultured for 18 h with cytokines in the absence or presence of ␣7 nAChR agonist or antagonist. To assess NKG2D-dependent cytotoxicity, we used a mouse urothelial carcinoma cell (MB49) transduced to stably express MICA on the cell surface (one of the known ligands for NKG2D (36,37)) or its corresponding negative control transduced with empty vector as target cells (Fig. 6A). We observed that NK cells display a higher cytotoxic activity against MB49-MICA cells than against MB49-pMSCV (negative control) cells, confirming the role of the NKG2D-MICA receptor-ligand pair in this response (Fig. 6B). However, NK cells exposed previously to the specific ␣7 nAChR agonist display a significantly reduced cytotoxic activity against MB-49-MICA cells, close to the background cytotoxicity observed with MB49-pMSCV cells (Fig. 6B). We conclude that PNU-282987 blunts NKG2D-dependent cytotoxicity of NK cells, which is in line with the observed down-regulation of NKG2D induced by the ␣7 nAChR agonist. Similarly, the percentage of IFN-␥-producing NK cells is significantly lower if NK cells are stimulated with cytokines and exposed to PNU-282987 (Fig. 6C). In both functional assays, the effect of PNU-282987 is prevented by ␣-BTX (Fig. 6, B and C), indicating that ␣7 nAChR negatively regulates NKG2D-dependent cytotoxicity and IFN-␥ production of NK cells. To gain further insight into the biological consequences of ␣7 nAChR activation on human NK cells and its repercussion on their cross-talk with DCs, human NK cells were stimulated for 48 h with IL-12/IL-18/IL-15, and further cultured for 18 h with cytokines in the absence or presence of PNU-282987. Thereafter, these NK cells were co-cultured with immature DCs (iDCs) for 4 h and we evaluated the expression of MHC-II, CD86 (a costimulatory molecule), and CD83 (DC maturation marker) on DCs (Fig. 7). We observed that pre-treatment of cytokine-stimulated NK cells with PNU-282987 results in significantly lower expression of MHC-II and CD83 in DCs, and less percentage of CD86 high DCs (Fig. 7) compared with cytokine-stimulated NK cells not exposed to the ␣7 nAChR agonist. These data indicate that activation of ␣7 nAChR on NK cells attenuates DC activation/maturation.
␣7 nAChR Stimulation Reduces Expression and Nuclear Mobilization of NF-B-Stimulation of NK cells with cytokines triggers intracellular pathways, some of which converge in NF-B activation. Besides, activation of ␣7 nAChR inhibits NF-B activation in macrophages (38). Therefore, we explored whether activation of this transcription factor is affected by ␣7 nAChR activation in human NK cells stimulated with IL-12, IL-18, and IL-15 (Fig. 8). We observed that NK cells stimulated with cytokines display an intense nuclear staining and that PNU-282987 produces a reduction in the nuclear mobilization of NF-B p65 (Fig. 8, A, B, and D). This effect is abolished if cells are preincubated 15 min with ␣-BTX (Fig. 8C). In addition, the expression of NF-B p65 is lower in cytokine-stimulated NK cells exposed to PNU-282987 than in those not exposed to the agonist as assessed by both fluorescence microscopy (Fig. 8D) and FC (Fig. 8, E and F).

Discussion
The present study is, to our knowledge, the first to demonstrate the presence of functional ␣7 nAChR on human NK cells, which is up-regulated after cytokine stimulation. We show that ␣7 nAChR activation in cytokine-stimulated NK cells triggers Ca 2ϩ mobilization from intracellular stores, decreases IFN-␥ production probably by reducing NF-B levels and its nuclear translocation, decreases cell-cytotoxic activity due to downregulation of NKG2D receptor, and attenuates the activation/ maturation of DCs.
Interestingly, NK cells from mouse do not express ␣7 nAChR mRNA (29). Such a difference in ␣7 nAChR expression between these two species has also been shown in lymphocytes (8,30,32,39,40), and may underlie a different neuroimmunomodulatory potential of lymphoid cells in them. In mouse, nicotine exposure impairs the ability of NK cells to kill target cells and release cytokines through ␤2-containing nAChRs (29). Because it has been proposed that ␤2 and ␣7 subunits contribute differently to the nicotine-dependent modulation of immune cell functions (41,42), it would be required to further explore the functional role of ␣4␤2 in human NK cells to understand the effects of nonspecific nAChRs ligands, such as nicotine. Our findings indicate that human NK cells can adjust to environmental changes because ␣7 nAChR is up-regulated by the presence of IL-12, IL-18, and IL-15, probably to control excessive IFN-␥ secretion and cytotoxicity that could induce damage in the surrounding healthy cells. This phenomenon is not unique to NK cells as it has been shown that stimulation of T cells alters the expression of nAChR subunits (8, 30 -32).
We show that specific activation of ␣7 nAChR mediates Ca 2ϩ signaling in NK cells. PNU-282987 causes a sustained increase in [Ca 2ϩ ] i in human NK cells, which is larger in the presence of a PAM. Similar types of responses were reported in other human non-neuronal cells, such as mesenchymal stem cells (43), endothelial cells (44), peripheral blood lymphocytes and leukemic cell lines (31,45), and platelets (46). The relatively slow response may be due to the fact that the release of Ca 2ϩ from intracellular stores is the underlying mechanism associated to ␣7 nAChR activation in these cells. This is in line with previous reports showing that activation of ␣7 nAChR leads to Ca 2ϩ mobilization from intracellular stores through a Ca 2ϩinduced Ca 2ϩ release mechanism, probably through inositol 1,4,5-trisphosphate and ryanodine receptors (6,47,48). Similar to our results, in lymphocyte T cells mobilization of Ca 2ϩ through the ␣7 nAChR channel is not necessarily required for the ␣7-induced release of Ca 2ϩ from the internal stores (45).
NK cells are crucial components of the immune response against tumors and virus-infected cells (49), and they play a key role in the regulation of the adaptive immune response (19,25). The stimulation of NK cells can occur through various cytokines, such as IL-12, IL-18, IL-2, IL-15, and type I IFNs (23,33), and through the engagement of activating receptors (23,26), triggering their effector functions. We found that activation of ␣7 nAChR in cytokine-stimulated NK cells decreases the expression of NKG2D, which constitutes the underlying mechanism for the observed decrease of the NKG2D-dependent cytotoxicity against MICA-expressing target cells. Activation of ␣7 nAChR also decreases the production of IFN-␥ in response to cytokines. To elucidate the mechanism underlying this effect, we measured NF-B because in macrophages it has been associated with the reduction in pro-inflammatory cytokine production, such as TNF-␣ (16, 38, 50). We found that in human stimulated NK cells, ␣7 nAChR activation leads to reduced levels and reduced nuclear translocation of NF-B p65, thus identifying one of the mechanisms underlying the antiinflammatory effect of ␣7 nAChR in these cells.
T-cell priming in vivo requires DCs maturation, which is in turn induced upon recognition of pathogen-derived molecular patterns as well as inflammatory cytokines (51-53). NK cells have been shown to contribute to DC maturation and in this manner, contribute to shift the adaptive immune response toward a Th1-biased profile (20 -22). As our results show that activation of ␣7 nAChR in NK cells impairs DCs maturation, we can speculate that this may impact on the ability of DCs cells to induce optimal activation and differentiation of T cells into Th1 cells during the course of the adaptive immune response against different pathogens or tumor cells (51)(52)(53).
Due to the high complexity and the multiple possible pathways involved, even in neuronal cells Ca 2ϩ signaling triggered by ␣7 nAChR has not been completely resolved (6). The mechanisms that link the early increase in cytosolic Ca 2ϩ with the final anti-inflammatory effects have not yet been completely elucidated (14, 54 -56). ␣7 nAChR has been shown to modulate several signaling pathways, such as JAK2/STAT3/NF-B, PLC/ inositol 1,4,5-trisphosphate, and PI3K/Akt/Nrf-2 pathways, which in immune cells result in potent anti-inflammatory effects through inflammatory cytokine production inhibition and overexpression of hemooxigenase-1 (54,(57)(58)(59). Also, a direct coupling of ␣7 nAChR to G-protein has been recently described (60,61). Thus, our study opens doors to decipher in human NK cells the highly complex signaling pathways triggered by ␣7 nAChR and their association to different cell functions.
Blood concentrations of ACh and choline are probably low to efficiently activate ␣7 nAChRs of immune cells (62)(63)(64)(65). However, immune cells, including T and B cells, macrophages, and DCs have been shown to be capable of synthesizing and releasing ACh, a process that is up-regulated during inflammation (66). Also, lymph nodes have an additional source of ACh from autonomic nerve terminals (67). Local levels of choline, another full agonist of ␣7 nAChR, can be elevated under conditions associated with ischemia, stroke, and substantial plasma mem-  brane damage (65,68). Therefore, it is likely that ACh and choline primarily act on the immune system by wiring transmission between close interactions between leukocytes or leukocyte and neurons (see reviews Refs. 65 and 69). Besides, immune cells also express SLURP-1 (secreted mammalian Ly-6/urokinase plasminogen activator receptor-related protein 1), a positive allosteric modulator of ␣7 nAChR (70,71). Moreover, SLURP-1 has also been detected in human blood and plasma (63,72). Accordingly, ␣7 nAChR is therefore emerging as an important drug target for the modulation of inflammation in different physiopathological contexts (12)(13)(14)(15)(16)(17). Thus, based on our results, NK cells may constitute an additional player of the cholinergic anti-inflammatory pathway.
Potentiation of ␣7 nAChR can be achieved by the use of agonists or PAMs, and both types of ligands emerge as potential therapeutic drugs (16,73,74). In particular, PAMs are of interest because they enhance ␣7 nAChR responses maintaining the temporal spatial characteristics of the endogenous activation, and they show higher specificity than agonists. We here show that PNU-120596 potentiates ␣7 nAChR responses in NK cells. Thus, ␣7 nAChR in NK cells could constitute another important drug target for immune modulation.
The anti-inflammatory effects of ␣7 nAChR signaling in NK cells observed in this study may constitute a physiological strategy to prevent or limit excessive tissue damage during the immune response against tumors and virus-infected cells. The present study opens the possibility to specifically modulate ␣7 nAChR activity as a novel pathway to regulate human NK cells.
Cell Lines-MB49-pMSCV or MB49-MICA were generated as follows. For retrovirus production, the retrovirus packaging cell line PT67 was transfected with viral DNA (pMSCV and pMSCV/MICA*008) and the packaging vectors (pCMVgag-pol and pMD2.G) using Lipofectamine Plus (Invitrogen). After 48 h, virus-containing supernatants were harvested, filtered, and used for infection as follows: 1 ml of viral supernatant containing PolyGram (8 mg/ml) was used to infect 5 ϫ RT-PCR Analysis-Total RNA was extracted with TRIzol reagent (Invitrogen Life Technologies) following the manufacturer's instructions. RNA was converted into cDNA using the Molony murine leukemia virus reverse transcriptase (Promega) and random primers (Promega). End point PCR was run for 40 cycles in a Mini Cycler (MJ Research). Real time qPCR was performed using a Rotor-Gene 6000 (Corbett Research). Amplification included 2 min at 94°C followed by 40 cycles of a three-step loop: 20 s at 94°C, 40 s at 56°C, and 40 s at 72°C. Results were expressed as the fold-increase of gene expression of cytokine-treated cells versus control (non-treated) cells. Results of gene expression were normalized against the 18S rRNA gene, the expression of which was not changed in stimulated cells. The 18S rRNA primer sequences used were: 5Ј-TCGAGGCCCTGTAATTGGAA-3Ј (sense) and 5Ј-CCCTC-CAATGGATCCTCGTT-3Ј (antisense); the ␣7 nAChR subunit primer sequences used were: 5Ј-CCAATGACTCGCAAC-CACTC-3Ј (sense) and 5Ј-GGTTCTTCTCATCCACGTCC-3Ј (antisense); the ␣4 nAChR subunit primer sequences used were: 5Ј-GGATGAGAAGAACCAGATGATG-3Ј (sense) and 5Ј-CTCGGAGGGGATGCGGAT-3Ј (antisense); and the ␤2 nAChR subunit primer sequences used were: 5Ј-CGGATACA-GAGGAGCGGC-3Ј (sense) and 5Ј-TGCACACTGATGAGC-TGG-3Ј (antisense). To avoid contamination of genomic DNA, the sense and antisense primers annealed to sequences in different exons, respectively.
Flow Cytometry-Cells were stained with fluorochromelabeled mAbs, analyzed in a FACSCalibur or a FACSAria flow cytometers (BD Biosciences), and data were processed with the FlowJo software (Tree Star Inc., Ashland, OR). Expression of cell surface receptors on NK cells was analyzed by FC as previ-ously described (75) and results were expressed as mean fluorescence intensity (MFI).
IFN-␥ Production by NK Cells-NK cells from healthy donors were stimulated for 48 h at 37°C with 10 ng/ml of IL-12, 10 ng/ml of IL-18, and 1 ng/ml of IL-15, were incubated for 18 h in the absence or presence of ␣7 nAChR-specific drugs. During the last 14 and 4 h of culture, GolgiPlug (BD) and GolgiStop (BD) were added, respectively. Cells were harvested and stained with anti-CD56 mAb, permeabilized with Cytofix/Cytoperm (BD Biosciences), and stained with the anti-IFN-␥ mAb to assess IFN␥-producing cells by FC.
NK Cell-mediated Cytotoxicity-NK cells from healthy donors were stimulated for 48 h at 37°C with 10 ng/ml of IL-12, 10 ng/ml of IL-18, and 1 ng/ml of IL-15, were incubated for 18 h in the absence or presence of ␣7 nAChR-specific drugs. Then, NK cells were washed and co-cultured with eFluor Dye 670labeled MB49-pMSCV or MB49-MICA cells at a 1:1 ratio. After 5 h, cells were labeled with Zombie Green and analyzed by FC. Percentage of cytotoxicity was calculated as 100ϫ percentage of eFluor Dye 670 ϩ Zombie Green high cells/percentage of eFluor Dye 670 ϩ cells (76).
Isolation and Culture of DCs-Monocytes were isolated from the blood of healthy volunteers provided by the Hemotherapy Unit of the Hospital Carlos Durand or the Service of Transfusion Medicine of the Hospital Churruca-Visca, both of Buenos Aires, Argentina, by immunomagnetic selection of CD14 ϩ cells using MACS (Miltenyi Biotech, Bergisch Gladbach, Germany). Purity of isolated cells was always above 90%, as assessed by FC as CD14 ϩ cells. Monocytes were cultured for 6 days with GM-CSF and IL-4 to obtain iDCs. These iDCs were cultured with NK cells (previously stimulated with cytokines in the absence or presence of ␣7 nAChR-specific ligands as described in the previous section) at a 1:1 ratio for 4 h in RPMI 1640 supplemented with 10% inactivated fetal bovine serum, sodium pyruvate, glutamine, and gentamicin. Thereafter, DCs were harvested and used for analysis of cell surface markers by FC.
␣-BTX Binding Assays-Cultured NK cells were harvested, washed, and incubated in the absence (total binding) or presence of 1 M ␣-BTX (nonspecific binding) for 30 min. Then, cells were incubated with Alexa Fluor 488-labeled ␣-BTX (1 g/ml) for 1 h. Thereafter, fluorescence was assessed by FC and fluorescence microscopy. For fluorescence microscopy, cells were mounted and analyzed by fluorescence microscopy using a Nikon Eclipse E-600 microscope. Images were obtained with a SBIG Astronomical Instrument (Santa Barbara, CA) provided with a CCDOPS software package (version 5.02) to drive a model ST-7 digital charge-coupled device camera. Appropriate dichroic and emission filters were used and images were analyzed using the ImageJ software (National Institutes of Health, Bethesda, MD).
Measurement of Changes in [Ca 2ϩ ] i -NK cells from healthy donors were stimulated for 48 h at 37°C with 10 ng/ml of IL-12, 10 ng/ml of IL-18, and 1 ng/ml of IL-15 and transferred to coverglass bottomed culture dishes coated with poly-L-lysine for 2 h (77) and incubated with 3 M of the Ca 2ϩ -sensitive fluorescent indicator Fluo-3/AM (Molecular Probes). [Ca 2ϩ ] i was assessed using a confocal laser scanning microscope (Leica DMIRE2). Fluo-3/AM-loaded cells were excited at 488 nm, and the fluorescence emission above 530 nm was acquired using a ϫ20 objective. Images of fluorescence were obtained at a rate of one frame every 4.2 s during 152.8 s. PNU-282987 (30 M) and PNU-120596 (3 M) were added directly to the samples. Basal fluorescence (F 0 ) was calculated prior to the addition of ␣7 nAChR ligands. Data were normalized to F 0 (F/F 0 ratio) to control for variations in basal fluorescence. The F/F 0 ratio was used to express the relative change in intracellular Ca 2ϩ over time.
Detection of NF-B-NK cells were stimulated for 48 h at 37°C with 10 ng/ml of IL-12, 10 ng/ml of IL-18, and 1 ng/ml of IL-15 and transferred to coverglass bottomed culture dishes coated with poly-L-lysine for 2 h. Then, NK were incubated for 2 h in the absence or presence of ␣7-nAChR specific ligands, fixed with 2% of PFA, permeabilized with 0.1% saponin for 10 min, and stained with anti-p65 mAb (sc-109, Santa Cruz Biotechnology, Santa Cruz, CA) and FITC-labeled goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories). Thereafter, NF-B was assessed by FC and fluorescence microscopy. For fluorescence microscopy, cells were mounted with Dabco, and observed in a confocal laser scanning microscope (Leica DMIRE2) using a ϫ60 objective. Fluorescence images were analyzed with the software ImageJ. For each cell, regions of interest were drawn around the nucleus and cytoplasm. The average fluorescence intensity for each regions of interest was measured from 8-bit images in at least 100 cells for each experimental condition.
Statistical Analysis-Data were plotted as mean Ϯ S.E. A one-way ANOVA test with Bonferroni post hoc test was used when three or more experimental groups were compared; a two-way ANOVA with repeated measures matched by both factors and Sidak post hoc test was used for the cytotoxicity experiment and a paired t test was used when two experimental groups were compared.