Cholinergic modulation of dendritic cell function

Cholinergic modulation of dendritic cell function

Journal of Neuroimmunology 236 (2011) 47–56 Contents lists available at ScienceDirect Journal of Neuroimmunology j o u r n a l h o m e p a g e : w w...

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Journal of Neuroimmunology 236 (2011) 47–56

Contents lists available at ScienceDirect

Journal of Neuroimmunology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j n e u r o i m

Cholinergic modulation of dendritic cell function☆ Gabriela Salamone a, b,⁎, Gabriela Lombardi c, Soledad Gori a, Karen Nahmod a, Carolina Jancic a, María Marta Amaral a, Mónica Vermeulen a, b, Alejandro Español c, María Elena Sales c, Jorge Geffner a, b a b c

Departamento de Inmunología, Instituto de Investigaciones Hematológicas and Instituto de Estudios Oncológicos “Fundación Maissa”; Academia Nacional de Medicina, Argentina Departamento de Microbiología, Parasitología e Inmunología, Facultad de Medicina, Universidad de Buenos Aires, Argentina Centro de Estudios Farmacológicos y Botánicos (CEFYBO)-CONICET, Facultad de Medicina, Universidad de Buenos Aires, Argentina

a r t i c l e

i n f o

Article history: Received 1 February 2011 Received in revised form 3 May 2011 Accepted 14 May 2011 Keywords: Dendritic cells Cholinergic system Muscarinic receptors Acetylcholine

a b s t r a c t Dendritic cells (DCs) are highly specialized antigen-presenting cells with a unique ability to activate resting T lymphocytes. Acetylcholine (ACh) is the primary parasympathetic neurotransmitter and also a non-neural paracrine factor produced by different cells. Here, we analyzed the expression of the cholinergic system in DCs. We found that DCs express the muscarinic receptors M3, M4 and M5, as well as the enzymes responsible for the synthesis and degradation of ACh, choline acetyltransferase (ChAT) and acetylcholinesterase (AChE), respectively. Differentiation of DCs in the presence of the cholinergic agonist carbachol, the synthetic analog of ACh, resulted in an increased expression of HLA-DR and CD86 and the stimulation of TNF-α and IL8 production. All these effects were prevented by atropine, a muscarinic ACh receptor (mAChR) antagonist. Carbachol, was also able to modulate the function of DCs when added after the differentiation is accomplished; it increased the expression of HLA-DR, improved the T cell priming ability of DCs, and stimulated the production of TNF-α but not IL-12 or IL-10. By contrast, carbachol significantly inhibited the stimulation of HLA-DR expression and the enhancement in the T cell priming ability of DCs triggered by LPS. Interestingly, the TNF-α antagonist etanercept completely prevented the increased expression of HLA-DR induced by carbachol, suggesting that it promotes the phenotypic maturation of DCs by stimulating the production of TNF-α. ACh induced similar effects than carbachol; it stimulated the expression of HLA-DR and the production of TNF-α, while inhibiting the stimulation of HLA-DR expression and IL-12 production triggered by LPS. Similarly, neostigmine, an inhibitor of AChE, also stimulated the expression of HLA-DR and the production of TNF-α by DCs while inhibiting the production of TNF-α and IL-12 triggered by LPS. These results support the existence of an autocrine/paracrine loop through which ACh modulates the function of DCs. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Conventional dendritic cells (DCs) are specialized antigen presenting cells with a unique ability to activate resting T cells and to direct their differentiation into different effector profiles (Guermonprez et al., 2002; Steinman, 2003; Ardavin et al., 2004; Reis e Sousa, 2006; Sabatte et al., 2007). It is well known that the nervous system and the immune system communicate bidirectionally, and that lymphoid tissues are innervated by the autonomic nervous system (Blalock and Weigent, 1994). Abbreviations: DCs, dendritic cells; ACh, acetylcholine; mAChR, muscarinic acetylcholine receptor; AChE, acetylcholinesterase; ChAT, choline acetyltransferase. ☆ This work was supported by grants from the Agencia Nacional de Promoción Científica y Tecnológica, Fundación Alberto Roemmers, and the Universidad de Buenos Aires (Argentina). ⁎ Corresponding author at: IIHEMA,Academia Nacional de Medicina, Pacheco de Melo 3081, 1425 Buenos Aires, Argentina. Tel.: + 54 11 4805 5759x296; fax: + 54 11 4803 9475. E-mail address: [email protected] (G. Salamone). 0165-5728/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2011.05.007

Moreover, increasing evidence suggests the presence of a nonneuronal cholinergic system in immunocompetent cells, which is activated during inflammation (Kurzen et al., 2007). In fact, the components of the cholinergic system; ACh, nicotinic and muscarinic receptors (n- and mAChRs), choline acetyltransferase (ChAT) and acetylcholinesterase (AChE), were detected in mammalian nonneuronal cells, including immune cells such as B and T lymphocytes (Kawashima and Fujii, 2000). Moreover, ACh was shown to be able to modulate the function of immune cells; it stimulates the activation of T CD8+ cells (Kawashima and Fujii, 2000; Zimring et al., 2005) while inhibits the production of inflammatory cytokines by phagocytes (Borovikova et al., 2000b; Pavlov and Tracey, 2005). There are not previous studies directed to analyze the expression of the cholinergic system in human DCs and the ability of ACh to modulate their functional profile. Here, we show that human DCs express the receptors M3, M4 and M5 as well as the enzymes responsible for the synthesis and degradation of ACh. Moreover, we found that the cholinergic agonist carbachol and ACh modulate the function of DCs. Interestingly, we found that cholinergic agonists

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induced opposite effects on resting and LPS-triggered DCs indicating that the ability of the cholinergic system to modulate the function profile of DCs is strongly dependent on their activation status.

Ethical Committee. All blood donors provided written informed consent for the collection of samples and subsequent analysis. 2.1. Reagents

2. Materials and methods The studies performed in this work have been reviewed and approved by the Academia Nacional de Medicina Review Board and

Endotoxin-free reagents and plastic materials were used in all experiments. RPMI-1640 and PBS were purchased from HyClone Laboratories (Logan, UT, USA). Fetal calf serum (FCS) and

Fig. 1. Human dendritic cells express the muscarinic receptors M3, M4 and M5. Dendritic cells were obtained from human monocytes cultured with IL-4 plus GM-CSF for 5 days. To obtain mature DCs, they were treated for 24 h with 100 ng/ml of LPS. The expression of muscarinic receptors was evaluated by western-blot (a and b), and confocal microscopy (c). (a) Upper panel: expression of muscarinic receptors in untreated and LPS-treated DCs. Lower panel: different cell types were used as negative (−) and positive (+) controls for the expression of the different muscarinic receptors, as described under Materials and methods. A representative experiment (n = 5) is shown in (a) and (c). Data quantified by densitometry are shown in (b). These data are expressed as the mean ± SEM of 5 experiments.

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penicillin/streptomycin were purchased from Invitrogen Life Technologies (Grand Island, NY, USA). Recombinant human interleukin-4 (IL-4) was from BD Pharmingen (San Diego, CA). Lipopolysaccharide (LPS) from Escherichia coli, recombinant human granulocyte–macrophage colony-stimulating factor (GM-CSF), ACh, carbachol and mecamylamine (MM), were from Sigma-Aldrich (St. Louis, MO). Neostigmine was from Laboratory Phadapharma, Buenos Aires, Argentina, atropine (AT) was from Laboratory Larjan-Buenos Aires, Agentina, TNF-α was from SigmaAldrich and etanercept was from Wyeth, Buenos Aires, Argentina.

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stained with the following mAbs: FITC- or PE-conjugated mAbs directed to CD1a, CD40, CD86, HLA-DR, CD83 and CCR7 (BD Pharmingen, San Diego, CA). The expression of muscarinic receptors was evaluated using specific goat or rabbit IgG polyclonal antibodies directed to the muscarinic receptors M1, M2, M3, M4, or M5 (Santa Cruz Biotech, Germany) and secondary FITC-labeled polyclonal IgG antibodies directed to goat or rabbit IgG (Sigma-Aldrich). Analysis was performed by using a FACS flow cytometer and CellQuest software (BD Biosciences, San Jose, CA).

2.2. Blood samples 2.5. Western blotting Blood samples were obtained from healthy nonsmoker men donors. Blood was obtained by venipuncture of the forearm vein, and it was drawn directly into heparinized plastic tubes. 2.3. Generation of human DCs Peripheral blood mononuclear cells (PBMC) were isolated from peripheral blood by Ficoll-Hypaque (1.077 g) density gradient centrifugation. CD14 + cells were then isolated by positive selection according to the manufacturer's instructions Miltenyi Biotec. (Germany). The purity was checked by FACS analysis using anti-CD14 monoclonal antibody (mAb) and was found to be N95%. To obtain DCs, monocytes (106/ml) were cultured in RPMI 1640 medium supplemented with 10% of heat-inactivated FCS, 50 U/ml penicillin, 50 μg/ml streptomycin, 20 ng/ml IL-4, and 20 ng/ml GM-CSF. On day 5, the cells were analyzed by FACS. To evaluate the effect of cholinergic agonists or antagonists on the differentiation of DCs, cell cultures were supplemented with drugs every two days. 2.4. Flow cytometry Cells were washed twice with PBS supplemented with 2% FCS and suspended in PBS supplemented with 10% heat-inactivated FCS. Fluorescein isothyocyanate (FITC) and phycoerythin (PE) conjugated mAbs were added at saturating concentrations for 30 min at 4 °C, and two additional washes were then performed. Human cells were

DCs (2× 10 6) were cultured with or without LPS (100 ng/ml) during 24 h at 37 °C. Then, cells were washed by the addition of 1 ml ice-cold saline supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF). The pellets were immediately frozen in dry ice after aspiration of the supernatants. Cell pellets were resuspended in loading buffer (2% SDS, 10% glycerol, 5% 2 β-mercaptoethanol and trace amounts of bromophenol blue dye in 62.5 mM Tris–HCl, pH 6.8), heated for 5 min at 95 °C and stored at −70 °C. Samples were then separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes (Sigma-Aldrich), and then blocked with 5% bovine albumin in PBS containing 0.05% Tween 20. Membranes were then blotted with the indicated antibodies overnight at 4 °C. Specific bands were developed by ECL (Amersham Biosciences). The antibodies used were: goat polyclonal IgG antimuscarinic receptors M1, M2, or M3, rabbit polyclonal IgG anti-M4 or M5 (Santa Cruz Biotech, Germany), mouse IgG mAb anti-ChAT (Chemicon Internat., Canada)and goat polyclonal IgG anti-AChE (Santa Cruz). Secondary polyclonal IgG antibodies directed to goat, rabbit, and mouse IgG labeled with HRP (Sigma-Aldrich), were also used. As a negative control for the expression of M1, M2 and M5 receptors the breast cancer cell line MCF-7 was employed. Neutrophils were used as negative controls for the expression of M3 and M4 receptors. The T cell line Jurkat was used as a positive control for the expression of M1 and M2 receptors, the breast cancer cell line MCF-7 as a positive control for M3 and M4 receptors, and human peripheral blood lymphocytes as a positive control for M5 receptors.

Fig. 2. Analysis of the expression of muscarinic receptors in dendritic cells by flow cytometry. The expression of muscarinic receptors in untreated and LPS-treated DCs was analyzed by flow cytometry, as described in Materials and methods. A representative experiment (n = 3) is shown.

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2.6. Confocal microscopy

2.7. RT-PCR

Immunostaining was done on resting (immature) and LPS-treated DCs. Primary antibodies were applied in Tris-buffered saline (TBS) with 3% heat-treated donkey serum and 0.25% Triton X-100. The antibodies used were: goat polyclonal IgG anti-muscarinic receptors M1, M2, or M3, rabbit polyclonal IgG anti-muscarinic receptors M4 or M5 (Santa Cruz), mouse IgG mAb anti-ChAT, and IgG mAb anti-AChE (Chemicon). Secondary antibodies were donkey anti-mouse cyanine 2 (Cy2), donkey anti-rabbit Cy3, donkey anti-goat Cy5, and donkey antimouse Cy3, (Jackson ImmunoResearch, West Grove, PA). All analyses were performed using a Zeiss LSM confocal microscope [LSM510Axiovert 100 M (Carl Zeiss, Gottingen, Germany)]. Images of labeled DCs were acquired using an optical slice of 1 μm interval with a plane aprochromatic 63× oil immersion objective [C-Apochromat 633/1.2 W objective, and a C-Apochromat 403/1.2 W objective lens]. Expression of markers and localization were done on 30 cells per slice.

Total RNA was extracted from human DCs using TRIzol reagent (Invitrogen Life Technologies). The reverse-transcription reaction was done using 3 μg of total RNA, in the presence of M-MLV reverse transcriptase enzyme (Promega). Forward and reverse primers for ChAT were: sense: 5′-GGAGATGTTCTGCTGCTATG; antisense: 3′GGAGGTGAAACCTAGTGGCA (280 pb), as previously described (Song et al., 2003). PCR products were separated on a 1.5% agarose gel, stained with ethidium bromide, and visualized by an UV transilluminator. 2.8. Mixed lymphocyte reaction (MLR) Dendritic cells were cultured for 24 h with or without carbachol or LPS. Cells were fixed in glutaraldehyde 0.001% (Sigma-Aldrich) for 30 s and washed three times in PBS/glycine 0.2 M. DCs (1 × 10 4/100 μl) were cultured alone or in the presence of 2 × 10 5

Fig. 3. Human dendritic cells express the enzymes choline acetyltransferase (ChAT) and acetylcholinesterase (AChE). The expression of ChAT in untreated and LPS (100 ng/ml)treated DCs was analyzed by PCR (a), western-blot (b), and confocal microscopy (f), while the expression of AChE was analyzed by western-blot (d), and confocal microscopy (g). Representative experiments are shown in figures a, b, d, f and g (n = 4–5). Data quantified by densitometry are shown in (c) and (e). These data are expressed as the mean ± SEM of 4–5 experiments. *p b 0.05 for DCs + LPS vs DCs.

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freshly isolated allogeneic PBMCs during 5 days in 96-well U-bottom plates (Costar). Thymidine incorporation was measured on day 5 by a 16-h pulse with [ 3H]thymidine (1 μCi/well, specific activity, 5 Ci/mM; DuPont).

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2.9. Measurement of cytokine production Cytokines (IL-8, IL-12p70, IL-10, TNF-α, IL-6 and MCP-1) in cell supernatants were measured by ELISA (R&D Systems).

Fig. 4. Differentiation of dendritic cells in the presence of the cholinergic agonist carbachol increases the expression of HLA-DR and CD86. DCs were obtained from human monocytes cultured with IL-4 and GM-CSF for 5 days. Cultures were performed with or without carbachol (10− 9 M), in the absence or presence of atropine (AT, 10− 7 M) or mecamylamine (MM, 10− 7 M). Then, the phenotype of DCs was analyzed by flow cytometry. Data showed in (a) and (c) represent the mean fluorescence intensity ± SEM (n = 5, *p b 0.05 for carbachol vs untreated). Histograms from representative experiments are shown in (b), (d) and (e).

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2.10. Statistical analysis Statistical significance was determined using the nonparametric Friedman test for multiple comparisons with Dunn's posttest or Student's t test. Statistical significance was defined as p b 0.05.

Fig. 1a–c shows that resting and LPS-treated DCs express the muscarinic receptors M3, M4 and M5, but not the M1 and M2 receptors. Similar results were observed when the expression of mAChR was analyzed by flow cytometry (Fig. 2). A large fraction of DCs express mAChR and no differences were observed when the expression of mAChR was analyzed in resting vs LPS-activated DCs.

3. Results 3.1. Human dendritic cells express the muscarinic receptors M3, M4 and M5

3.2. Human dendritic cells express the enzymes choline acetyltransferase and acetylcholinesterase

The expression of mAChR has been previously reported in lymphocytes (Kawashima et al., 1998). We here analyzed the expression of these receptors in DCs. In a first set of experiments, we studied whether immature and mature human DCs express mAChR. DCs were obtained from human monocytes cultured for 5 days in the presence of GM-CSF and IL-4. Maturation of DCs was induced by LPS (100 ng/ml, 24 h). In all cases, the expression of mAChR was evaluated by western-blot and confocal microscopy.

The expression of the enzymes ChAT and AChE, responsible for the synthesis and degradation of ACh, was analyzed in immature and mature DCs. Dendritic cells were obtained from human monocytes cultured with IL-4 and GM-CSF for 5 days. Mature DCs were obtained after stimulation of immature DCs for 24 h with LPS (100 ng/ml). Assays performed by PCR, western-blot and confocal microscopy showed that immature and mature DCs express ChAT (Fig. 3a–c, and f). The expression of ChAT was found to be lower in LPS-treated DCs

Fig. 5. Differentiation of dendritic cells in the presence of the cholinergic agonist carbachol stimulates the production of TNF-α and IL-8. DCs were obtained from human monocytes cultured with IL-4 and GM-CSF for 5 days. Cultures were performed with or without carbachol (10− 9 M), in the absence or presence of atropine (AT, 10− 7 M) or mecamylamine (MM, 10− 7 M). Then, the production of TNF-α, IL-8, IL-12p70, IL-10, IL-6, and MCP-1 was analyzed by ELISA. Data are expressed in pg/ml and represent the mean ± SEM of 6 experiments (*p b 0.05 for carbachol vs untreated).

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compared with resting DCs (Fig. 3c). Moreover, assays performed by western-blot and confocal microscopy showed that DCs also express AChE (Fig. 3d, e and g). Similar levels of AChE expression was observed in both, immature and mature DCs (Fig. 3e). 3.3. The cholinergic agonist carbachol modulates the differential profile of dendritic cells DCs were obtained from human monocytes cultured with IL-4 and GM-CSF for 5 days, in the absence or presence of carbachol. Then, the phenotype of DCs was analyzed by flow cytometry, and the production of cytokines was determined by ELISA. Fig. 4a–d show that carbachol, at a concentration of 10− 9 M, significantly increased the expression of HLADR and CD86. Similar effects were induced using 10− 7 M and 10− 8 M of carbachol, while no effects were observed using 10 − 10 M of carbachol (data not shown). Carbachol was completely unable to modulate the expression of CD1a, CD40, CD83, and CCR7 in DCs (Fig. 4e). The stimulatory effect of carbachol on the expression of HLA-DR and CD86 was completely abrogated by the mAChR antagonist atropine (AT), while the nicotinic antagonist mecamylamine (MM) did not exert any effect. Moreover, Fig. 5 shows that carbachol (10− 9 M) significantly increased the production of TNF-α and IL-8 by DCs (Fig. 5a and b). This effect was completely prevented by AT but not by MM. No effect of carbachol on the production of IL-12p70, IL-10, IL-6 and MCP-1 was detected (Fig. 5c–f). 3.4. Cholinergic agonists modulate the function of dendritic cells when added after the differentiation is accomplished In these experiments, DCs were differentiated from monocytes cultured for 5 days with IL-4 and GM-CSF, in the absence of cholinergic

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agonists. Then, we analyzed the ability of carbachol to modulate the phenotype and function of DCs. Culture of DCs with carbachol during 24 h resulted in the up-regulation of HLA-DR (Fig. 6a and b). Of note, carbachol significantly prevented the up-regulation of HLA-DR expression induced by LPS (Fig. 6a and b). These changes in the expression of HLA-DR correlated with T cell-priming capacity, as indicated by the ability of carbachol to enhance the MLR response mediated by DCs cultured without LPS and to prevent the increase in the MLR response mediated by LPS-treated DCs (Fig. 6c). We also analyzed the ability of carbachol to modulate the production of the proinflammatory cytokines TNF-α and IL-12 by DCs. Carbachol stimulated the production of TNF-α without modifying the production of IL-12 (Fig. 7a and b). Consistent with the observations made for HLA-DR expression, we found that carbachol significantly prevented the stimulation of TNF-α and IL12p70 production by LPS-triggered DCs (Fig. 7a and b). Because TNF-α is able to induce the phenotypic maturation of DCs (Guermonprez et al., 2002; Sabatte et al., 2007), we then analyzed whether the stimulation of TNF-α production by carbachol might account for its ability to stimulate the expression of HLA-DR in DCs. Etanercept, a TNF-α antagonist, completely prevented the up-regulation of HLA-DR expression induced by carbachol (Fig. 7c), suggesting that it increases the expression of HLADR by stimulating the production of TNF-α in an autocrine and/or paracrine way. We then analyzed whether ACh was able to mediate similar effects than carbachol. The results obtained are shown in Fig. 8. Similar than carbachol, ACh induced contrasting effects on resting and LPSactivated DCs. It stimulated the expression of HLA-DR and the production of TNF-α by DCs cultured in the absence of LPS, while significantly prevented the up-regulation in the expression of HLA-DR and the increased production of TNF-α and IL-12 induced by LPS.

Fig. 6. Carbachol modulates the phenotype and function of dendritic cells. DCs were obtained from human monocytes cultured with IL-4 and GM-CSF for 5 days. Then, they were cultured for 24 h with or without LPS (100 ng/ml), in the absence or presence of carbachol (10− 7 to 10− 9M). The expression of HLA-DR by DCs was analyzed by flow cytometry (a and b) and the ability of DCs to mediate a mixed lymphocyte reaction (MLR) was assessed as described under Materials and methods using DCs (1 × 104/100 μl) and 2 × 105 freshly isolated allogeneic PBMCs cultured for 5 days in 96-well U-bottom plates (c). (a) Histograms from a representative experiment (n = 5) are shown. (b) The results are expressed as the percentage of increase in the mean fluorescence intensity of HLA-DR in carbachol-, LPS-, or carbachol plus LPS-treated DCs compared with untreated DCs (mean ± SEM, *p b 0.05 for carbachol vs untreated and LPS plus carbachol vs LPS). (c) Results are expressed as cpm and represent the mean ± SEM of 5 experiments performed in triplicate. (**p ≤ 0.01 carbachol vs untreated, and *p ≤ 0.05 carbachol plus LPS vs LPS). The values of cpm for DCs or allogeneic PBMCs cultured alone were in all cases lower than 1000.

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Fig. 7. Carbachol modulates the production of TNF-α and IL-12 by dendritic cells. (a and b) DCs were obtained from human monocytes cultured with IL-4 and GM-CSF for 5 days. Then, they were cultured for 24 h with or without LPS (100 ng/ml), in the absence or presence of carbachol (10− 7 to 10− 9M). The production of TNF-α and IL-12p70 was then evaluated by ELISA. Data are expressed in pg/ml and represent the mean ± SEM of 5–7 experiments (*p b 0.05 carbachol vs untreated, and carbachol plus LPS vs LPS). (c) DCs, obtained as described above, were cultured for 24 h with TNF-α (50 ng/ml) or carbachol (10−8M), in the absence or presence of etanercept (0.5 μM). Then, the expression of HLA-DR was analyzed by flow cytometry. Histograms from a representative experiment (n = 3) are shown.

3.5. The acetylcholinesterase inhibitor neostigmine modulates the function of dendritic cells Having shown that DCs express the enzymes responsible for the synthesis and degradation of ACh (ChAT and AChE, respectively), and to explore a possible role of endogenous ACh in the control of DC function, we performed a new set of experiments using the AChE inhibitor, neostigmine. DCs were differentiated from human monocytes cultured for 5 days with IL-4 and GM-CSF. Then, they were cultured for 24 h, in the absence or presence of LPS, with or without neostigmine, and the expression of HLA-DR and the production of TNF-α and IL-12p70 were assessed. Fig. 9 shows that neostigmine exerts similar effects than those induced by cholinergic stimuli. It increased the expression of HLA-DR and stimulated the production of TNF-α by resting DCs, while significantly preventing the production of TNF-α and IL-12 p70 triggered by LPS. However, and contrasting with the effects induced by cholinergic stimuli, neostigmine was completely unable to prevent the up-regulation of HLA-DR expression triggered by LPS. 4. Discussion Our results indicate that human monocyte-derived DCs express the muscarinic receptors M3, M4 and M5, as well as the enzymes responsible for the synthesis and degradation of ACh, ChAT and AChE, respectively. Moreover, we found that cholinergic stimulation during the differentiation of DCs increases the expression of HLA-DR and CD86 and stimulates the production of TNF-α and IL-8, acting through mAChR. Similar results were observed when cholinergic agonists were added after the differentiation of DCs was accomplished.

Moreover, the ability of the TNF-α antagonist etanercept to prevent the increased expression of HLA-DR induced by carbachol suggests that cholinergic stimulation increases the expression of HLA-DR by stimulating the production of TNF-α in an autocrine and/or paracrine way. Of note, contrasting effects were observed when cholinergic stimuli were added to LPS-triggered DCs instead of resting DCs; they partially prevented the stimulation of HLA-DR expression and the production of TNF-α and IL-12 triggered by LPS. Together, our results suggest that the effect of cholinergic stimulation on DC function is strongly dependent on their maturation status. Interestingly, the ability of the AChE inhibitor neostigmine to reproduce all these effects suggests the existence of an autocrine/paracrine loop through which the endogenous production of ACh might be able to modulate the function of DCs. Our results are in line with the so called cholinergic antiinflammatory pathway, first appreciated by Borovikova et al. (Borovikova et al., 2000a). This model supports that the stimulation of the vagus nerve and the consequent release of ACh suppress the production of TNF-α in LPS-challenged rats, inhibiting the development of shock. This effect appears to involve an anti-inflammatory action exerted by ACh on spleen macrophages, perhaps induced through the homopentameric α7 nicotinic acetylcholine receptor (Tracey, 2009). Similar anti-inflammatory effects were induced by cholinergic stimulation in different experimental models of pancreatitis (van Westerloo et al., 2006), dextran sulfate sodiuminduced colitis (Ghia et al., 2006), and ischemia–reperfusion injury (Bernik et al., 2002). Previous studies have also analyzed whether immune cells have a functional cholinergic system. It has been shown that T cells express all known mAChR (M1 to M5), various subtypes of nAChR and the

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Fig. 8. Acetylcholine modulates the phenotype and function of dendritic cells. DCs were obtained from human monocytes cultured with IL-4 and GM-CSF for 5 days. Then, they were cultured with or without LPS (100 ng/ml), in the absence or presence of ACh (10−7 to 10−9M). The expression of HLA-DR by DCs was analyzed by flow cytometry and the production of TNF-α and IL-12p70 was measured by ELISA. (a) Histograms from a representative experiment (n = 5) are shown. (b) The results are expressed as the percentage of increase in the mean fluorescence intensity of HLA-DR in ACh-, LPS-, or ACh plus LPS-treated DCs compared with untreated DCs (*p b 0.05 ACh vs untreated and ACh plus LPS vs LPS). (c and d) Data are expressed in pg/ml and represent the mean ± SEM of 4–6 experiments (*p b 0.05 ACh vs untreated and ACh plus LPS vs LPS).

enzyme responsible for the synthesis of ACh; ChAT (Fujii et al., 1999). Macrophages also express some nAChR (Galvis et al., 2006) and it has been shown that stimulation of the nAChR in peritoneal macrophages increased phagocytosis and suppressed the production of inflammatory cytokines triggered by LPS (van der Zanden et al., 2009). Regarding DCs, murine bone-marrow derived DCs have shown to express different nAChR. On the other hand, Aicher et al.(2003) observed that human monocyte-derived DCs express the α7 nAChR. Moreover, supporting an anti-inflammatory function of nAChR in human DCs, it has been shown that nicotine decreased the production of inflammatory cytokines (IL-1β, TNF-α, and IL-12) triggered by LPS (Nouri-Shirazi and Guinet, 2003). Interestingly, other studies have shown that nicotine, acting on resting human DCs, stimulates not only the expression of co-stimulatory molecules but also the production of IL-12 and their ability to stimulate T-cell proliferation (Aicher et al., 2003); (Gao et al., 2007). Consistent with our present data, these results reinforce the notion that cholinergic stimulation might be able to exert different effects on DCs depending on their maturation status. Recently, Liu et al.(2010), investigated the role of mAChR in nasal mucosal immune cells. The authors found that DCs isolated from surgically removed nasal mucosa express the muscarinic receptor M3. Moreover, when cultured with the muscarinic agonist metacholine they found a marked increase in the production of OX40L by DCs. This

molecule interacts with OX40, a member of the TNF receptor family expressed by activated T cells. It has been shown that OX40-mediated signals promote the activation of inflammatory responses by both, CD4+ and CD8+ T cells while inhibiting the function of natural Foxp3+ T regulatory cells. Thus, the results from Liu et al. suggest that cholinergic stimulation of DCs through muscarinic receptors improves the inflammatory activity of DCs. Consistent with this presumption, we found that cholinergic stimulation during the differentiation of DCs results in the stimulation of the expression of HLA-DR and the production of TNF-α, and that both effects were abrogated by atropine. Together, our results support the notion that the function of DCs in peripheral tissues might be regulated by ACh produced by either neural or non-neural cells, including DCs themselves. Our results also suggest that ACh might induce opposite effects on DC function depending on their maturation status or the simultaneous presence of other stimuli such as pathogen-associated molecular patterns (PAMPs), cytokines or chemokines.

Acknowledgements We thank Mabel Horvat and Beatriz Loria for their valuable assistance.

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Fig. 9. The acetylcholinesterase inhibitor neostigmine modulates the function of dendritic cells. DCs were obtained from human monocytes cultured with IL-4 and GMCSF for 5 days. Then, they were cultured for 24 h, in the absence or presence of LPS (100 ng/ml), with or without neostigmine (20 μM). The expression of HLA-DR was evaluated by flow cytometry (a) and the production of TNF-α and IL-12p70 was analyzed by ELISA (b and c). (a) A representative experiment (n = 4) is shown. (b and c) Data are expressed in pg/ml and represent the mean ± SEM of 3–4 experiments (*p b 0.05 NEO vs untreated and NEO plus LPS vs LPS).

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