Although noradrenaline (NA), a stress-associated neurotransmitter, seems to affect the immune system, the precise mechanisms underlying NA-mediated immunoregulation are not fully understood. We examined the effect of NA on Ag uptake (endocytosis) by dendritic cells (DCs) using murine bone marrow-derived DCs and fluorescence-labeled endocytic tracers (dextran and OVA). Ag uptake by DCs notably increased following a very brief treatment (3 min) with NA. NA-induced endocytosis was completely blocked by treatment with α2-adrenoceptor antagonist yohimbine. Neither α1-adrenoceptor antagonist prazosin nor β-adrenoceptor antagonist propranolol affected NA-induced endocytosis by DCs. A selective α2-adrenoceptor agonist, azepexole (B-HT 933), also significantly increased endocytosis by DCs. Thus, the α2-adrenoceptor seems to be responsible for NA-induced DC endocytosis. In parallel, NA markedly activated intracellular signaling pathways of PI3K and ERK1/2 in DCs. NA-mediated activation of these pathways was completely inhibited by yohimbine treatment. Blocking PI3K activation significantly reduced NA-induced endocytosis by DCs. Based on these results, NA rapidly enhances Ag capture by DCs via α2 adrenoceptor-mediated PI3K activation, which may be associated with immune enhancement following acute stress.

Stress plays a role in the pathogenesis and progression of various diseases (1). Several stressors induce immune alterations and, thereby, influence the susceptibility or severity of immune disorders, such as infection and allergy (2, 3). Chronic stress induces immune suppression, whereas acute stress may enhance some aspects of immune functions (4, 5). Indeed, brief amounts of stress (<2 h) were shown to augment delayed-type hypersensitivity skin responses following cutaneous Ag exposure (4). Although mechanisms underlying immune dysfunction upon chronic stress have been well documented, the immune enhancement following acute stress is less well characterized. The sympathetic nervous system is a major pathway responsible for stress-induced immune alterations (2, 3). Stress events activate the sympathetic nervous system and induce secretion of noradrenaline (NA), the main synaptic neurotransmitter. In addition to various nonlymphoid tissues, sympathetic nerve fibers extend to lymphoid organs, including thymus, spleen, lymph nodes, GALT, and bone marrow (6). NA released from the end of sympathetic nervous fibers in these lymphoid organs seems to alter the immune cell functions via adrenergic receptors (ARs).

Dendritic cells (DCs) are potent APCs and play a major role in the regulation of immune responses to a variety of Ags (79). It was reported that NA alters the balance of cytokine production by DCs upon TLR stimulation (1012). NA treatment for ≥3 h suppresses DC production of proinflammatory cytokines, including IL-12 and TNF-α, whereas it enhances the production of IL-10, an anti-inflammatory cytokine (1012), similarly to cAMP-elevating agents, such as PGE2 and forskolin (13). It seems that NA-mediated alteration of cytokine balance is involved, at least in part, in immune suppression upon chronic stress. The AR family is composed of α11A, α1B, and α1D), α22A, α2B, and α2C), and β (β1, β2, and β3) receptors, coupled with guanine nucleotide-binding protein (G protein) (1416). The effect of NA on cytokine production seems to be mainly mediated via β ARs coupled with Gs proteins responsible for the elevation of intracellular cAMP levels and the activation of protein kinase A (PKA) (1012). DCs were shown to express β ARs, as well as α1 and α2 ARs. It was reported that α1 ARs are involved in DC migration to the lymph nodes in mice (17). However, the role of α2 ARs in DC functions and α2 AR-mediated signal transduction in DCs remains to be elucidated.

DCs circulate through lymphoid organs, as well as almost all tissues as sentinels in the immune surveillance system (79). DCs first capture Ags via endocytosis from extracellular fluid following injury and subsequent invasion of pathogens and then present these Ags in the context of MHC class II molecules at the cell surface to activate the Ag-specific CD4+ T cells. In parallel, the sympathetic nervous system seems to release NA in response to injury (18), whereas NA signaling seems to be quickly terminated by diffusion, reuptake, and degradation of this transmitter.

Although DC endocytosis of Ags is essential to induce acquired immunity, the precise mechanisms underlying the regulation of this process are not fully understood. We examined the effect of short-term NA exposure (3–20 min) on the functions of DCs using murine bone marrow-derived DCs (BMDCs). In this article, we demonstrate that NA induces DC endocytosis via α2 AR signaling within a very short time (3 min). We also demonstrate involvement of the PI3K pathway in α2 AR-mediated endocytosis of DCs.

RPMI 1640 liquid medium was purchased from Sigma-Aldrich (St. Louis, MO) and supplemented with 100 IU/ml penicillin and 100 μg/ml streptomycin. Murine rGM-CSF was obtained from PeproTech (Rocky Hill, NJ). Rabbit complement (Low Tox-M) was acquired from Cedarlane Laboratories (Hornby, Ontario, Canada). Dextran (10,000 m.w.) conjugated with Alexa Fluor 488 (A488-dextran) and OVA conjugated with Alexa Fluor 488 (A488-OVA) were acquired from Molecular Probes (Eugene, OR). Con A conjugated with rhodamine (Rho-ConA) was obtained from Vector Laboratories (Burlingame, CA). NA, prazosin (α1 AR-specific antagonist), yohimbine (α2 AR-specific antagonist), propranolol (β AR-specific antagonist), azepexole (B-HT933, α2 AR-specific agonist), LY294002 (a specific inhibitor of PI3K), and H89 (a specific inhibitor of PKA) were purchased from Sigma-Aldrich. U0126, a specific inhibitor of MEK1/2, was purchased from Calbiochem (San Diego, CA). LY294002, H89, and U0126 were used at 10 μM, based on prior studies (19, 20) and our preliminary dose-response study. Anti–phospho-Akt (Ser473) Ab, anti–phospho-ERK1/2 (Thr202/Tyr204) Ab, anti–phospho-p38 MAPK (Thr180/Tyr182) Ab, and anti-GAPDH mAb (14C10) were purchased from Cell Signaling Technology (Beverly, MA). LPS (ultrapure grade, Escherichia coli O111:B4) and Pam3CSK4 (P3C), a synthetic lipopeptide, were obtained from InvivoGen (San Diego, CA).

Murine BMDCs were generated as previously described (21, 22). Bone marrow cells were prepared from femur and tibial bone marrow of C57BL/6 mice (Japan SLC, Hamamatsu, Japan). After lysis of erythrocytes, MHC class II+, CD45R+ (B220), CD4+, and CD8+ cells were removed by killing with mAbs (1E4, RA3-6B2, GK1.5, and 53.4.9) and rabbit complement. The cells were cultured in RPMI 1640 medium containing 5% FCS, 20 ng/ml GM-CSF, and 50 μM 2-ME at a density of 1 × 106 cells/ml/well using a 24-well plate. On day 2, the medium was gently exchanged with fresh medium. On day 4, nonadherent granulocytes were removed without dislodging clusters of developing DCs, and fresh medium was added. On day 6, free-floating and loosely adherent cells were collected and used as BMDCs (93–97% CD11c+B220).

The endocytosis assay was performed as previously described (23, 24). BMDCs (0.5–1 × 106 cells/ml) were incubated in 5% FCS RPMI 1640 buffered with 10 mM HEPES at 37°C. The endocytic tracer (A488-dextran or A488-OVA) and NA were added concurrently, prior to a 3–20-min incubation. A488-dextran or A488-OVA was added to a final concentration of 50 or 100 μg/ml, respectively. Endocytosis of the tracer was halted at the indicated time points by rapid cooling of the cells on ice. The cells were then washed with ice-cold PBS. The fluorescence intensity of the cells was analyzed by flow cytometry on a FACSAria (BD Biosciences, San Jose, CA). Incubation of cells with the endocytic tracer on ice was used as a background control. The mean fluorescence intensity (MFI) resulting from the subtraction of background control from each experimental sample represented the amount of incorporated tracer. To examine the effects of inhibitors on endocytosis, cells were pretreated for 30–60 min with each inhibitor at 37°C. After inhibitor pretreatment, the cells were incubated with the endocytic tracer and NA in the presence of the inhibitor.

To morphologically analyze endocytosis, cells (1.5 × 106 cells/ml, 400 μl) were incubated in 5% FCS RPMI 1640 buffered with 10 mM HEPES at 37°C. The endocytic tracer and NA were added concurrently, prior to 3 min of incubation. Rapid cooling of the cells on ice halted endocytosis of the tracer. Paraformaldehyde (4%, 1 ml) was added to the cell suspension, and the suspension was incubated for 10 min on ice to fix the cells. Following washing, the cells were stained with Rho-ConA. The cells were washed and resuspended in PBS. The cells were applied to a glass slide, and a coverslip was placed. Cell morphology and fluorescence intensity were analyzed by confocal microscopy (Nikon, Tokyo, Japan).

Immunoblotting was performed as previously described (25). BMDCs were treated with NA for 3, 7, or 15 min at a density of 1 × 106 cells/ml. To examine the effects of inhibitors, cells were pretreated for 60 min with each antagonist or inhibitor at 37°C. After inhibitor pretreatment, the cells were treated with NA at 37°C in the presence of the inhibitor. Rapid cooling of the cells on ice halted the reactions, and these cells were washed with ice-cold PBS. The whole-cell lysates were prepared using cell lysis buffer (Cell Signaling Technology). The cell lysates were separated by SDS-PAGE and then blotted onto a polyvinylidene fluoride membrane (Millipore, Bedford, MA). The membrane was probed with the primary Ab and developed with HRP-conjugated secondary Ab by ECL.

BMDCs were treated with LPS and P3C for 24 h in 5% FCS RPMI 1640 at a density of 1 × 106 cells/ml. In some experiments, cells were pretreated with an inhibitor for 60 min and then treated with LPS plus P3C for 24 h in the presence of the inhibitor. The culture supernatants were subjected to quantification of the protein level of IL-12 (p70) and IL-10 by ELISA. The ELISA kits for IL-12 (p70) and IL-10 were purchased from BD Biosciences.

The unpaired Student t test was used to analyze data for significant differences; p values <0.05 were regarded as significant.

We examined the effect of NA on DC endocytosis using a fluorescence-labeled dextran, a common endocytic tracer of DCs (23).BMDCs were incubated with A488-dextran in the presence or absence (control) of 1 μM NA for a variable time period, ranging from 3–20 min. Fluorescence intensity of the cells was analyzed by flow cytometry (Fig. 1). In the absence of NA, slight uptake of A488-dextran by DCs was detected 3 min after the tracer addition (Fig. 1A, upper panel) and then the fluorescence intensity of the cells was gradually increased at 5–20 min (Fig. 1B). Of note, NA notably increased A488-dextran uptake by DCs at 3 min (Fig. 1A [lower panel], Fig. 1B). Significant enhancement of A488-dextran uptake by DCs was also detected at 5–20 min after the NA treatment (Fig. 1B). Endocytosis of A488-dextran by DCs increased with the addition of NA in a dose-dependent manner, peaking at 1 μM after 3 min of treatment (data not shown).

FIGURE 1.

The effects of NA on the endocytosis of A488-dextran by DCs. BMDCs were incubated with A488-dextran in the presence or absence (control) of 1 μM NA for the indicated times. Fluorescence intensity of the cells was analyzed by flow cytometry. A, Data at 3 min, which are representative of three independent experiments. B, Time course of endocytosis in the presence of NA. MFI of the cells is shown. Each symbol represents the mean ± SE of three independent experiments. Statistical significance was calculated using the unpaired Student t test. **p < 0.01 versus control.

FIGURE 1.

The effects of NA on the endocytosis of A488-dextran by DCs. BMDCs were incubated with A488-dextran in the presence or absence (control) of 1 μM NA for the indicated times. Fluorescence intensity of the cells was analyzed by flow cytometry. A, Data at 3 min, which are representative of three independent experiments. B, Time course of endocytosis in the presence of NA. MFI of the cells is shown. Each symbol represents the mean ± SE of three independent experiments. Statistical significance was calculated using the unpaired Student t test. **p < 0.01 versus control.

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We next analyzed the morphology of NA-induced endocytosis using confocal microscopy (Fig. 2). BMDCs were incubated with A488-dextran in the presence or absence (control) of 1 μM NA for 3 min, and Rho-ConA was used to stain the cell surface. A488+ spots (green) were observed in the inside of the cell surface (red) in untreated (control) and NA-treated cells. NA treatment obviously increased the number of A488+ spots, corresponding to A488-dextran–incorporating endosomes, compared with those observed in control cells. Thus, NA increased A488-dextran incorporation into the endosomes within 3 min. Almost no merged area was detected in either group of cells. Consequently, fluorescence intensity of the cells in the analysis by flow cytometry reflects the amount of A488-dextran inside the cell but not adherent to the cell surface.

FIGURE 2.

Morphological evidence for NA-induced endocytosis. BMDCs were incubated with A488-dextran in the presence or absence (control) of 1 μM NA for 3 min. Following fixation and Rho-ConA staining (cell-surface staining), the fluorescence intensity of the cells was analyzed by confocal microscopy. A, Endocytosis in the presence or absence of NA; green, A488-dextran; red, Rho-ConA (original magnification ×40). B, Magnified image of NA-treated cells: A488 image (a), Rho image (b), and merged image (c) (original magnification ×40). Data are representative of three independent experiments.

FIGURE 2.

Morphological evidence for NA-induced endocytosis. BMDCs were incubated with A488-dextran in the presence or absence (control) of 1 μM NA for 3 min. Following fixation and Rho-ConA staining (cell-surface staining), the fluorescence intensity of the cells was analyzed by confocal microscopy. A, Endocytosis in the presence or absence of NA; green, A488-dextran; red, Rho-ConA (original magnification ×40). B, Magnified image of NA-treated cells: A488 image (a), Rho image (b), and merged image (c) (original magnification ×40). Data are representative of three independent experiments.

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BMDCs were shown to express α1, α2, and β ARs (26). We then examined which receptors were responsible for NA-induced endocytosis in DCs. BMDCs were pretreated with α1-adrenergic antagonist prazosin (1 μM), α2-adrenergic antagonist yohimbine (1 μM), or β-adrenergic antagonist propranolol (1 μM) and then incubated with A488-dextran and 1 μM NA for 5 min. Fluorescence intensity of the cells was analyzed by flow cytometry (Fig. 3A, 3B). NA again increased A488-dextran uptake by DCs. NA-induced endocytosis was unaffected by treatment with prazosin or propranolol. In contrast, yohimbine treatment completely blocked the effect of NA on DC endocytosis. Yohimbine showed no effect on spontaneous DC endocytosis.

FIGURE 3.

Involvement of α2 ARs in NA-induced endocytosis by DCs. BMDCs were pretreated with 1 μM prazosin (Pra), 1 μM yohimbine (Yoh), or 1 μM propranolol (Pro) and then incubated with A488-dextran (A, B) or A488-OVA (C, D) in the presence of 1 μM NA or 10 μM azepexole for 5 min (A, B) or 3 min (C, D). Fluorescence intensity of the cells was analyzed by flow cytometry. A and C, A representative graph of three independent experiments is shown. B and D, MFI of the cells is shown. Each column represents the mean ± SE of three independent experiments. Statistical significance was calculated using the unpaired Student t test. *p < 0.05 versus NA alone; **p < 0.01 versus untreated control.

FIGURE 3.

Involvement of α2 ARs in NA-induced endocytosis by DCs. BMDCs were pretreated with 1 μM prazosin (Pra), 1 μM yohimbine (Yoh), or 1 μM propranolol (Pro) and then incubated with A488-dextran (A, B) or A488-OVA (C, D) in the presence of 1 μM NA or 10 μM azepexole for 5 min (A, B) or 3 min (C, D). Fluorescence intensity of the cells was analyzed by flow cytometry. A and C, A representative graph of three independent experiments is shown. B and D, MFI of the cells is shown. Each column represents the mean ± SE of three independent experiments. Statistical significance was calculated using the unpaired Student t test. *p < 0.05 versus NA alone; **p < 0.01 versus untreated control.

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We also examined DC uptake of A488-OVA, a representative protein Ag, upon NA exposure. BMDCs were incubated with A488-OVA in the presence of 1 μM NA or 10 μM azepexole (B-HT933), the α2-AR specific agonist, for 3 min (Fig. 3C, 3D). NA notably increased A488-OVA uptake by DCs. Azepexole also significantly increased A488-OVA uptake by DCs. In the presence of yohimbine, neither NA nor azepexole increased DC endocytosis of A488-OVA. Yohimbine showed no effect on spontaneous A488-OVA uptake by DCs. Thus, α2 ARs seemed to be responsible for the enhancement of DC endocytosis upon NA treatment.

It was reported that NA induces activation of Akt and ERK1/2 in PC12 cells stably expressing each of the three human α2-AR subtypes but not in wild-type cells (27). However, to the best of our knowledge, no study has reported activation of these molecules in DCs following NA exposure. We next examined the effect of NA treatment on activation of Akt (a downstream effector of PI3K), ERK1/2, and p38 MAPK in DCs. BMDCs were treated with 1 μM NA for 3, 7, or 15 min, and the intracellular protein levels of the active forms of these molecules, p-Akt, p-ERK1/2, and phospho-p38 MAPK (pp38), were determined by immunoblotting (Fig. 4A). Low levels of p-Akt, p-ERK1/2, and pp38 were detected in untreated DCs. The level of p-Akt was greatly increased 3 min after NA treatment. The increased level was maintained at 7 and 15 min after NA treatment. The level of p-ERK1/2 also increased 3 min after NA treatment. The p-ERK1/2 level in NA-treated DCs decreased at 7 and 15 min compared with the level at 3 min. In contrast, NA showed little or no effect on the pp38 levels.

FIGURE 4.

Activation of Akt and ERK1/2 in DCs upon NA treatment. A, Kinetics of Akt and ERK1/2 activation. BMDCs were incubated with 1 μM NA for the indicated times. The intracellular protein levels of p-Akt, p-ERK1/2, pp38, and GAPDH were determined by immunoblotting. B and C, The effects of AR antagonists on Akt and ERK1/2 activation. BMDCs were pretreated with 1 μM prazosin, 1 μM yohimbine, or 1 μM propranolol and then incubated with 1 μM NA for 3 min in the presence of each inhibitor. A and B, A representative immunoblot from three independent experiments is shown. C, The relative intensity of the specific band is shown. Each column represents the mean ± SE of three independent experiments. Statistical significance was calculated using the unpaired Student t test. *p < 0.05, **p < 0.01 versus NA alone.

FIGURE 4.

Activation of Akt and ERK1/2 in DCs upon NA treatment. A, Kinetics of Akt and ERK1/2 activation. BMDCs were incubated with 1 μM NA for the indicated times. The intracellular protein levels of p-Akt, p-ERK1/2, pp38, and GAPDH were determined by immunoblotting. B and C, The effects of AR antagonists on Akt and ERK1/2 activation. BMDCs were pretreated with 1 μM prazosin, 1 μM yohimbine, or 1 μM propranolol and then incubated with 1 μM NA for 3 min in the presence of each inhibitor. A and B, A representative immunoblot from three independent experiments is shown. C, The relative intensity of the specific band is shown. Each column represents the mean ± SE of three independent experiments. Statistical significance was calculated using the unpaired Student t test. *p < 0.05, **p < 0.01 versus NA alone.

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We next investigated which receptors were responsible for the activation of Akt and ERK1/2 in DCs. BMDCs were pretreated with prazosin, yohimbine, or propranolol and then treated with NA for 3 min (Fig. 4B, 4C). Prazosin slightly or partially diminished the level of p-Akt or p-ERK1/2 in DCs upon NA treatment. Notably, yohimbine almost completely blocked the NA-mediated increase in the levels of p-Akt and p-ERK1/2. In contrast, propranolol showed no significant effect on the levels of p-Akt and p-ERK1/2. Thus, α2 ARs seemed to be responsible for Akt and ERK1/2 activation in DCs following NA exposure.

To explore the role of PI3K/Akt and ERK1/2 activation following NA exposure, we examined the effect of LY294002, a specific inhibitor of PI3K, and U0126, a specific inhibitor of MEK1/2 (the upstream activator of ERK1/2), on NA-induced endocytosis in DCs. BMDCs were pretreated with LY294002 or U0126 and then treated with 1 μM NA in the presence of each inhibitor for 3 min. The levels of p-Akt and p-ERK1/2 were determined by immunoblotting (Fig. 5A, 5B). LY294002 completely blocked Akt activation upon NA treatment. LY294002 slightly decreased ERK1/2 activation upon NA treatment, although this effect was not statistically significant. In contrast, U0126 completely blocked activation of ERK1/2 upon NA treatment, while showing no effect on Akt activation.

FIGURE 5.

The effect of PI3K or ERK1/2 inhibition on DC endocytosis upon NA treatment. A and B, The effects of PI3K and MEK1/2 inhibitors on Akt and ERK1/2 activation. BMDCs were pretreated with 10 μM LY294002 (LY), a PI3K-specific inhibitor, or 10 μM U0126 (U), a MEK1/2-specific inhibitor, and then incubated with 1 μM NA for 3 min in the presence of each inhibitor. The intracellular protein levels of p-Akt, p-ERK1/2, and GAPDH were determined by immunoblotting. A, A representative immunoblot of three independent experiments is shown. B, The relative intensity of the specific band is shown. C and D, The effects of PI3K and MEK1/2 inhibitors on DC endocytosis. BMDCs were pretreated with 10 μM LY294002 (LY) or 10 μM U0126 (U) and then incubated with A488-dextran and 1 μM NA for 5 min. Fluorescence intensity of the cells was analyzed by flow cytometry. C, Representative graph of three independent experiments. D, MFI of the cells. Each column represents the mean ± SE of three independent experiments. Statistical significance was calculated using the unpaired Student t test. *p < 0.05, **p < 0.01 versus NA alone.

FIGURE 5.

The effect of PI3K or ERK1/2 inhibition on DC endocytosis upon NA treatment. A and B, The effects of PI3K and MEK1/2 inhibitors on Akt and ERK1/2 activation. BMDCs were pretreated with 10 μM LY294002 (LY), a PI3K-specific inhibitor, or 10 μM U0126 (U), a MEK1/2-specific inhibitor, and then incubated with 1 μM NA for 3 min in the presence of each inhibitor. The intracellular protein levels of p-Akt, p-ERK1/2, and GAPDH were determined by immunoblotting. A, A representative immunoblot of three independent experiments is shown. B, The relative intensity of the specific band is shown. C and D, The effects of PI3K and MEK1/2 inhibitors on DC endocytosis. BMDCs were pretreated with 10 μM LY294002 (LY) or 10 μM U0126 (U) and then incubated with A488-dextran and 1 μM NA for 5 min. Fluorescence intensity of the cells was analyzed by flow cytometry. C, Representative graph of three independent experiments. D, MFI of the cells. Each column represents the mean ± SE of three independent experiments. Statistical significance was calculated using the unpaired Student t test. *p < 0.05, **p < 0.01 versus NA alone.

Close modal

We next analyzed the effect of these inhibitors on DC endocytosis in response to NA. BMDCs were pretreated with LY294002 or U0126 and then incubated with A488-dextran and 1 μM NA for 5 min in the presence of each inhibitor. Fluorescence intensity of the cells was analyzed by flow cytometry (Fig. 5C, 5D). LY294002 treatment significantly diminished the NA-induced increase in DC endocytosis, while showing no significant effect on spontaneous DC endocytosis. In contrast, U0126 did not affect spontaneous or NA-induced endocytosis by DCs. Thus, the PI3K pathway, but not the ERK1/2 pathway, seemed to drive NA-mediated DC endocytosis.

It was reported that the endocytic capacity of DCs transiently increased following short-term treatment (30–40 min) with TLR ligands, such as LPS (28). We then examined the influence of LPS treatment on NA-induced endocytosis by DCs. BMDCs were pretreated with LPS for 30 min and then incubated with A488-dextran and NA in the presence of LPS for 3 min (Fig. 6). Similar to previous studies, LPS treatment increased DC uptake of A488-dextran. Of note, NA induced a further increase in endocytosis by DCs upon LPS stimulation. In the presence of yohimbine, NA failed to exert a significant effect on DC endocytosis upon LPS stimulation. Yohimbine alone showed no effect on DC endocytosis upon LPS stimulation. Thus, NA could increase Ag uptake of DCs upon LPS stimulation via α2 ARs.

FIGURE 6.

The effect of LPS on NA-induced endocytosis by DCs. BMDCs were pretreated with 1 μg/ml LPS with or without 1 μM yohimbine (Yoh) for 30 min and then incubated with A488-dextran and 1 μM NA for 3 min in the presence of LPS, with or without yohimbine. Fluorescence intensity of the cells was analyzed by flow cytometry. A, Representative graph from three independent experiments. B, MFI of the cells. Each column represents the mean ± SE of three independent experiments. Statistical significance was calculated using the unpaired Student t test. **p < 0.01.

FIGURE 6.

The effect of LPS on NA-induced endocytosis by DCs. BMDCs were pretreated with 1 μg/ml LPS with or without 1 μM yohimbine (Yoh) for 30 min and then incubated with A488-dextran and 1 μM NA for 3 min in the presence of LPS, with or without yohimbine. Fluorescence intensity of the cells was analyzed by flow cytometry. A, Representative graph from three independent experiments. B, MFI of the cells. Each column represents the mean ± SE of three independent experiments. Statistical significance was calculated using the unpaired Student t test. **p < 0.01.

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It was shown that NA altered the balance of cytokine production in DCs (1012). Although β AR-mediated regulation is well established, the role of α2 AR-mediated signaling in cytokine production is less consistent. Thus, we examined the influence of NA and the involvement of ARs in IL-12 and IL-10 production by DCs upon TLR stimulation. BMDCs were treated with 1 μM NA and TLR ligands (LPS plus P3C) for 24 h, and cytokine levels in culture supernatant were determined by ELISA. TLR ligands induced IL-12 and IL-10 production by DCs (Fig. 7A). NA completely inhibited IL-12 production by DCs upon TLR stimulation. On the contrary, NA increased IL-10 production by DCs. Prazosin and yohimbine showed no effect on NA-mediated alteration of IL-12 and IL-10 production upon TLR stimulation (Fig. 7B). In contrast, propranolol restored the decreased level of IL-12 production in NA-treated DCs, whereas it inhibited the NA-mediated increase in IL-10 production. Thus, the NA-induced alteration of cytokine production by DCs upon TLR stimulation seemed to be mediated via β ARs but not α1 and α2 ARs.

FIGURE 7.

The effect of NA on cytokine production by DCs. A, BMDCs were treated with LPS plus P3C (TLR-L) for 24 h in the presence or absence (−) of 1 μM NA. B, BMDCs were pretreated with 1 μM prazosin, 1 μM yohimbine, or 1 μM propranolol and then treated with TLR-L for 24 h in the presence or absence (−) of 1 μM NA. C, BMDCs were pretreated with 10 μM H89 and then treated with TLR-L for 24 h in the presence or absence (−) of 1 μM NA. The amounts of IL-12 and IL-10 in the culture supernatants were quantified by ELISA. Each column represents the mean ± SE of three independent experiments. Each column represents the mean ± SE of three (B, C) or four (A) independent experiments. Statistical significance was calculated using the unpaired Student t test. *p < 0.05; ** p < 0.01.

FIGURE 7.

The effect of NA on cytokine production by DCs. A, BMDCs were treated with LPS plus P3C (TLR-L) for 24 h in the presence or absence (−) of 1 μM NA. B, BMDCs were pretreated with 1 μM prazosin, 1 μM yohimbine, or 1 μM propranolol and then treated with TLR-L for 24 h in the presence or absence (−) of 1 μM NA. C, BMDCs were pretreated with 10 μM H89 and then treated with TLR-L for 24 h in the presence or absence (−) of 1 μM NA. The amounts of IL-12 and IL-10 in the culture supernatants were quantified by ELISA. Each column represents the mean ± SE of three independent experiments. Each column represents the mean ± SE of three (B, C) or four (A) independent experiments. Statistical significance was calculated using the unpaired Student t test. *p < 0.05; ** p < 0.01.

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The β ARs are coupled with G proteins responsible for intracellular cAMP elevation and subsequent PKA activation (16). We next examined the involvement of PKA in NA-mediated alteration of cytokine production by DCs using a PKA-specific inhibitor H89 (Fig. 7C). H89 treatment improved the decrease in IL-12 production caused by NA-treated DCs, whereas it inhibited the NA-mediated increase in IL-10 production. Thus, β AR-mediated PKA activation seemed to be responsible for the NA-mediated alteration of cytokine production by DCs upon TLR stimulation, whereas α2 AR-mediated signaling was unlikely to affect cytokine production.

The ARs are G protein-coupled receptors (GPCRs) that are composed of α1, α2, and β ARs that are coupled with Gq, Gi, and Gs proteins, respectively (1416). Although the effect of NA on cytokine production by DCs has been established (1012), the influence of this catecholamine on other DC functions is not well documented. It was reported that NA decreases IL-12 production and increases IL-10 production by DCs. NA-induced alteration of cytokine production seems to be mainly mediated via β ARs. The β AR pathway elevates intracellular cAMP level via Gs protein and, thereby, activates PKA (16). PKA activation seems to induce inhibition of IL-12 production and enhancement of IL-10 production in DCs (13). As noted in previous studies, NA modified the balance of IL-12 and IL-10 production by DCs upon TLR stimulation, and β-AR antagonist or PKA inhibition blocked this effect (Fig. 7). It seems that the signal cascade via β ARs, Gs protein, cAMP, and PKA is responsible for the NA-mediated alteration of cytokine production in DCs. In contrast, the role of α2 AR signaling in the immune system is less characterized. In addition to the classical roles of α2 ARs, recent studies showed that α2 AR signaling alters dendritic spine generation in neurons (29, 30). This effect might be responsible for synaptic modification during learning and memory in the central nervous system. The α2 ARs seem to be involved in reconstitution of the cytoskeleton in neurons. In the current study, we found that α2 AR signaling enhanced Ag uptake by DCs following a very short treatment with NA.

The α2 ARs are Gi protein-associated GPCRs and consist of three highly homologous subtypes: α2A-, α2B-, and α2C-ARs. BMDCs were shown to highly express α2A-AR but not α2B- or α2C-ARs (26), suggesting that NA-induced endocytosis is mainly mediated via α2A AR in BMDCs. In the current study, α2 AR-mediated signaling induced PI3K activation and rapid endocytosis in DCs, and blocking PI3K activation inhibited NA-mediated endocytosis. PI3Ks are divided into class I, class II, and class III, based on their structural similarities (31, 32). Class I PI3Ks control many cellular functions, including growth, proliferation, survival, adhesion, and migration. Class I PI3Ks are further divided into class IA (PI3Kα, PI3Kβ, and PI3Kδ) and class IB (PI3Kγ). Of note, PI3Kγ is mainly expressed by immune cells and activated by Gi protein-associated GPCRs, such as chemokine receptors (31). Taken together, we conclude that α2-AR/Gi-protein signaling and subsequent PI3Kγ activation induces DC endocytosis following NA stimulation.

It was reported that PI3K activation upon TLR stimulation is involved in the regulation of cytokine production by DCs (33). Thus, there was a possibility that α2 AR-mediated PI3K activation was also involved in the NA-mediated regulation of cytokine production upon TLR stimulation. However, inhibition of the α2-AR pathway, which completely blocked NA-mediated PI3K activation, showed no effect on the NA-mediated alteration of cytokine production. Therefore, NA-mediated PI3K activation is unlikely to be involved in NA-mediated cytokine regulation. It was shown that TLRs activate class IA PI3Ks, whereas GPCRs drive class IB PI3K (PI3Kγ) (31). Thus, the role of α2 AR-mediated PI3K activation seems to be different from that of TLR-mediated PI3K activation in the regulation of DC functions.

Although NA induced significant activation of ERK1/2 via α2 ARs in DCs, blocking the ERK1/2 pathway had no effect on endocytosis by NA-treated DCs (Fig. 5). Thus, the role of ERK1/2 activation in response to NA remains to be elucidated. We are exploring the role of α2 AR-mediated ERK1/2 activation in DC functions.

It was reported that NA suppressed phagocytosis of macrophages via α and β ARs (34). In contrast, some studies showed that NA increased phagocytosis of macrophages, although its effect was modest (35). Therefore, the effect of NA on endocytic activity in macrophages remains controversial. In the previous studies, NA treatment and phagocytosis assay were performed for ≥30 min. To the best of our knowledge, no previous report has shown regulation of endocytosis by NA in DCs. In the current study, we examined the effects of a short treatment (3–20 min) with NA on DC endocytosis and showed that NA notably induced rapid endocytosis via an α2 AR-mediated mechanism in DCs.

In the nervous system, signal transduction is controlled by spatial and temporal regulation of transmitter release, and signaling is terminated by diffusion, rapid reuptake, and degradation. This brief regulation system could apply for the immune system. NA exerted an effect on DC endocytosis within a very short time (3 min). Thus, upon acute stress, brief NA signaling from the sympathetic nervous system might augment Ag capture by DCs to enhance immune responses. It was suggested that very brief stress enhances some aspect of immune function (2). Our present findings may explain, at least in part, the immune enhancement upon acute stress.

The sympathetic nervous system seems to release NA following injury (18). In parallel, the injured tissue could be exposed to pathogenic microbes. It is physiologically possible that DCs are simultaneously exposed to NA and microbe components following injury. TLRs recognize pathogen-associated molecular patterns in the pathogen-derived molecules and are responsible for induction of innate immune responses (36). It was reported that DC facilitates increased Ag uptake following short exposure (30–40 min) to TLR ligands, such as LPS, but not after long-term stimulation (28). The LPS-induced transient enhancement of Ag capture by DCs seems to augment acquired immunity. In agreement with the previous study, brief treatment with LPS enhanced Ag capture by DCs (Fig. 6). In addition, we found that NA induced further enhancement of Ag capture by the LPS-treated DCs. It seems that LPS and NA cooperatively act on the Ag capture by DCs following injury and the subsequent invasion of pathogens.

The relationship between the endocytosis effect and the cytokine effect is an important point to consider with regard to the physiological role of NA in immune responses. Our present findings suggest that NA may induce endocytosis by DCs in peripheral tissue within a few minutes following a stress. Subsequently, DCs are activated by maturational stimuli, such as TLR ligands, and leave the peripheral tissue to migrate into the T cell area of the draining lymph nodes in several hours (37). The NA-mediated alteration in cytokine production by DCs upon TLR stimulation may affect DC-induced T cell polarization in the lymph nodes, because the effect of NA on cytokine production was detected several hours after NA treatment with TLR ligands (1012). The cytokine effect might have occurred in T cells and DCs during the Ag presentation in the lymph nodes. It seems that the endocytosis effect or the cytokine effect might be responsible for the quantity or quality of the immune responses, respectively.

In contrast, brief stress may induce temporal release of NA, resulting in short-term exposure of DCs to NA. In contrast, chronic stress may induce prolonged release of NA, resulting in long-term exposure of DCs to NA. Thus, we hypothesize that brief stress may induce the endocytosis effect only, whereas chronic stress may induce the endocytosis effect and the cytokine effect. This hypothesis seems to be compatible with previous studies showing that brief stress enhances some aspects of immune functions, whereas chronic stress induces immune suppression (4, 5).

In this study, we demonstrated a role for NA in the regulation of Ag capture by DCs. Because the endocytosis of exogenous Ags by DCs is essential for Ag presentation to T cells and the induction of adaptive immunity against the microorganisms, further elucidation of the mechanism underlying the NA-mediated regulation of DC endocytosis will lead to the development of clinical applications exploiting this regulation system for the treatment of various infectious diseases and immune disorders.

Disclosures The authors have no financial conflicts of interest.

Abbreviations used in this paper:

A488-dextran

dextran conjugated with Alexa Fluor 488

A488-OVA

OVA conjugated with Alexa Fluor 488

AR

adrenergic receptor

BMDC

bone-marrow derived dendritic cell

DC

dendritic cell

GPCR

G protein-coupled receptor

G protein

guanine nucleotide-binding protein

LY

LY294002

MFI

mean fluorescence intensity

NA

noradrenaline

P3C

Pam3CSK4

PKA

protein kinase A

pp38

phospho-p38 MAPK

Pra

prazosin

Pro

propranolol

Rho-ConA

concanavalin A conjugated with rhodamine

TLR-L

LPS plus Pam3CSK4

U

U0126

Yoh

yohimbine.

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