of March 31, 2018.This information is current as

through Neurokinin-2 ReceptorCell-Mediated Type 1 Immune Responses Neuropeptide Signaling Activates Dendritic

Takashi NishimuraHidemitsu Kitamura, Minoru Kobayashi, Daiko Wakita and

ol.1102521http://www.jimmunol.org/content/early/2012/04/02/jimmun

published online 2 April 2012J Immunol 

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The Journal of Immunology

Neuropeptide Signaling Activates Dendritic Cell-MediatedType 1 Immune Responses through Neurokinin-2 Receptor

Hidemitsu Kitamura,* Minoru Kobayashi,* Daiko Wakita,* and Takashi Nishimura*,†

Neurokinin A (NKA), a neurotransmitter distributed in the central and peripheral nervous system, strictly controls vital

responses, such as airway contraction, by intracellular signaling through neurokinin-2 receptor (NK2R). However, the function

of NKA–NK2R signaling on involvement in immune responses is less-well defined. We demonstrate that NK2R-mediated neuro-

peptide signaling activates dendritic cell (DC)-mediated type 1 immune responses. IFN-g stimulation significantly induced NK2R

mRNA and remarkably enhanced surface protein expression levels of bone marrow-derived DCs. In addition, the DC-mediated

NKA production level was significantly elevated after IFN-g stimulation in vivo and in vitro. We found that NKA treatment

induced type 1 IFN mRNA expressions in DCs. Transduction of NK2R into DCs augmented the expression level of surface MHC

class II and promoted Ag-specific IL-2 production by CD4+ T cells after NKA stimulation. Furthermore, blockade of NK2R by an

antagonist significantly suppressed IFN-g production by both CD4+ T and CD8+ T cells stimulated with the Ag-loaded DCs.

Finally, we confirmed that stimulation with IFN-g or TLR3 ligand (polyinosinic-polycytidylic acid) significantly induced both

NK2R mRNA and surface protein expression of human PBMC-derived DCs, as well as enhanced human TAC1 mRNA, which

encodes NKA and Substance P. Thus, these findings indicate that NK2R-dependent neuropeptide signaling regulates Ag-specific

T cell responses via activation of DC function, suggesting that the NKA–NK2R cascade would be a promising target in chronic

inflammation caused by excessive type 1-dominant immunity. The Journal of Immunology, 2012, 188: 000–000.

Asthma, affecting .300 million people worldwide, isa major public health problem; symptoms include re-current wheezing, coughing, and shortness of breath.

Generally, bronchial asthma, characterized as a Th2 cell-mediatedairway inflammation, causes elevation of serum IgE level, eosin-ophilia in the lung, and airway hyperresponsiveness (AHR) (1–3).However, some patients with asthma occasionally develop severeneutrophilia-like chronic obstructive pulmonary disease (COPD),and the pathology shows steroid drug resistance (4–8). The precisemechanisms of severe asthma, compared with classic allergicasthma, have not been elucidated.We established Th1-, Th2-, and Th17-dependent asthma models

by adoptive transfer of Ag-specific Th1, Th2, or Th17 cells intowild-type mice (9, 10). Then, we found that the Th2-mediatedasthma model was steroid sensitive, but the Th1 and Th17 mod-

els were steroid resistant, suggesting that both Th1 and Th17cause steroid-resistant severe airway inflammation. Furthermore,we performed intranasal injection with IFN-g and confirmed thedirect effect of IFN-g on the increased AHR (11). Then, wedemonstrated that the expression level of neurokinin-2 receptor(NK2R) was remarkably enhanced in the Th1-dependent asthmamodels and in the lung after IFN-g injection. In addition, we foundthat administration of NK2R antagonist significantly blocked theIFN-g–dependent increase in AHR. Therefore, these findingsstrongly suggest that NK2R-mediated neuropeptide signalingmight be closely related to IFN-g–induced airway inflammation.Substance P (SP) and neurokinin A (NKA), encoded by a single

TAC1 (Tac1) gene, are well-known neuropeptides, composed ofstructurally and evolutionarily conserved peptides characterizedby the common C-terminal sequence FXGLM-NH2. The precur-sor proteins of tachykinins, called preprotachykinins, are digestedinto smaller peptides by posttranslational proteolytic processing. Itwas demonstrated that they are located in the excitatory non-adrenergic and noncholinergic nerves of the mammalian respira-tory tract (12, 13). Therefore, release of tachykinins by theexcitation of these nerve systems is thought to be involved in thepathogenesis of airway allergy in humans (14, 15). Because thosefactors are produced in the airway tissues during inflammatoryresponses, developing therapy that blocks the tachykinin-depen-dent signaling cascade might be beneficial for the treatment ofallergic inflammation (16–18).Generally, SP secreted by neurons in response to local tissue

damage is capable of inducing and augmenting many inflammatoryresponses. Recent articles indicated that SP regulates the functionof various leukocytes, such as vascular endothelial growth factorsecretion from mast cells (19), cytotoxicity of NK cells (20), cy-tokine production by monocytes and T cells (21, 22), induction ofchemokine or chemokine receptor of macrophages (23, 24), andAg presentation of dendritic cells (DCs) (25). In contrast, NKA isknown to control various vital responses, such as airway con-traction, vasodilatation, and vascular permeability, in humans (15,

*Division of Immunoregulation, Section of Disease Control, Institute for GeneticMedicine, Hokkaido University, Sapporo 060-0815, Japan; and †Division of ROYCE’Health Bioscience, Section of Disease Control, Institute for Genetic Medicine, Hok-kaido University, Sapporo 060-0815, Japan

Received for publication September 2, 2011. Accepted for publication February 26,2012.

This work was supported in part by a Grant-in-Aid for a National Project “Knowl-edge Cluster Initiative” (second stage, “Sapporo Biocluster Bio-S”), a Grant-in Aidfor Scientific Research (B) (22300331 to T.N.), Grants-in Aid for Young Scientists(B) (22700894 to D.W. and 22790370 to H.K.) from the Ministry of Education,Culture, Sports, Science and Technology, Japan, and by the Joint Research Programof the Institute for Genetic Medicine, Hokkaido University.

Address correspondence and reprint requests to Dr. Takashi Nishimura, Division ofImmunoregulation, Institute for Genetic Medicine, Hokkaido University, Kita-15,Nishi-7, Kita-ku, Sapporo 060-0815, Japan. E-mail address: tak24@igm.hokudai.ac.jp

Abbreviations used in this article: 7AAD, 7-aminoactinomycin D; AHR, airwayhyperresponsiveness; BALF, bronchoalveolar lavage fluid; COPD, chronic obstruc-tive pulmonary disease; DC, dendritic cell; DT, diphtheria toxin; DTR, diphtheriatoxin receptor; GR, GR 159897; hNK2R, human neurokinin-2 receptor; hTAC1,human TAC1; NKA, neurokinin A; NK2R, neurokinin-2 receptor; poly I:C,polyinosinic-polycytidylic acid; SP, substance P; Tg, transgenic.

Copyright� 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00

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16, 26). However, the function of NKA in the immune system isless-well defined compared with the role of SP.The neurokinin receptors NK1R, NK2R, and NK3R encode

TACR1 (Tacr1), TACR2 (Tacr2), and TACR3 (Tacr3) genes inmammals, respectively. All of them belong to the rhodopsin-likefamily 1 of G protein-coupled receptors, which plays a key role incell signaling (27, 28). It is well known that SP exhibits prefer-ential binding to NK1R, which is expressed on immune cells, suchas monocyte and macrophages, in addition to cells of the nervoussystem. NKA has more affinity to NK2R, although naturally oc-curring tachykinins can stimulate any of the neurokinin recep-tors. Recent articles reported that NK2R expression was observedin neurons (29), murine macrophages (30), alveolar macrophagesfrom patients with COPD (31), tissue from chronic pancreatitis(32), and airway smooth muscle (33).In the present study, we first found that IFN-g stimulation

significantly enhanced surface NK2R expression levels on bothmurine and human DCs and then confirmed that DCs increase theNKA, a neuropeptide, level after IFN-g stimulation in vivo, aswell as in vitro. In contrast, NKA stimulation significantly inducedthe mRNA expression of type 1 IFN, IFN-a, and IFN-b in DCs.Finally, we demonstrated that the NKA–NK2R signaling cascadeactivates DC-mediated CD4+ T and CD8+ T cell responses. Fromthese findings, we propose a novel paradigm of NK2R-dependentcross-talk between the neural and immune systems in Th1/IFN-g–dependent chronic inflammation, including severe asthma.

Materials and MethodsMice

Wild-type BALB/c and C57BL/6 mice were purchased from Charles RiverJapan (Yokohama, Japan). Ifnar2/2 mice were kindly provided by Dr. Y.Iwakura (University of Tokyo, Tokyo, Japan). Stat12/2 mice were kindlyprovided by Dr. S. Akira (Osaka University, Osaka, Japan). CD11c-diphtheria toxin (DT) receptor (DTR) transgenic (Tg) mice were pur-chased from The Jackson Laboratory (Bar Harbor, ME). H-2b– and I-Ab–restricted OVA peptide-specific TCR Tg (OT-I and OT-II) mice wereprovided by F.R. Carbone (University of Melbourne, Victoria, Australia).All mice were maintained in specific pathogen-free conditions according tothe guidelines of our institute’s animal department and were used at 6–8wk of age.

Abs and reagents

Allophycocyanin-conjugated anti-CD11c mAb (HL3), PE-conjugated anti-CD11b mAb (M1/70), PE-conjugated anti–IFN-g mAb (XMG1.2), andPE-conjugated anti–I-Ab (AF6-120.1) were purchased from BD Biosci-ence (San Diego, CA). 7-aminoactinomycin D (7AAD) was purchasedfrom Beckman Coulter (Miami, FL). A rabbit polyclonal Ab against NK2R(M-48) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA),which reacted with NK2R of mouse, rat, and human origin. Control ratIgG was purchased from MP Biomedicals (Eschwege, Germany). Allo-phycocyanin-conjugated anti-human CD11c, FITC-conjugated HLA-DP/DQ/DR, and FITC-conjugated anti-human HLA-A/B/C were purchasedfrom BD Biosciences. Recombinant mouse IFN-g, murine GM-CSF, hu-man IFN-g, human GM-CSF, and human IL-4 were purchased fromPeproTech (Rocky Hill, NJ). LPS and DT were purchased from Sigma-Aldrich (St. Louis, MO). Polyinosinic-polycytidylic acid (poly I:C) waspurchased from InvivoGen (San Diego, CA). NKA agonist (NKA 140-07171) was purchased from Wako Chemical Industries (Osaka, Japan).NK2R selective antagonist GR 159897 (GR) was purchased from TocrisBioscience (Bristol, U.K.). GR was a potent selective antagonist of NK2Rand had negligible activity against NK1R or NK3R (34). The OT-I(chicken OVA257–264, SIINFEKL) and the OT-II (chicken OVA323–339, ISQAVHAAHAEINEAGR) peptides were kindly supplied by Dr. H.Tashiro (Fujiya, Hadano, Japan). OVA protein was purchased from Sigma-Aldrich Japan (Tokyo, Japan). pMX-IRES-GFP retrovirus vectors werekindly provided by Dr. T. Kitamura (University of Tokyo).

Generation of bone marrow-derived DCs

Bone marrow cells were prepared from wild-type, Ifna2/2, and Stat12/2

mice. DCs were generated from bone marrow cells with RPMI 1640

medium containing 10% FCS in the presence of murine GM-CSF (20 ng/ml) or the culture supernatant of GM-CSF–producing CHO cells, as de-scribed previously (35). Loosely adherent clustering cells were harvestedon days 6–8, and CD11c+ DCs were isolated by the IMag Cell SeparationSystem with anti-CD11c mAb-bound beads or FACSAria (BD Bio-sciences). The sorted CD11c+ DCs were immature (CD11chigh MHC classIIlow), and the purity was .95% (data not shown). These DCs were usedfor the present experiments. The generated DCs were treated with murineIFN-g (10 ng/ml), NKA (1 mM), or LPS (1 mg/ml) for 6 h and used for theevaluation of mRNA expression levels. For analysis of NK2R proteinexpression levels, DCs were treated with IFN-g (10 ng/ml) for 24 h. ForNKA assays, DCs were treated with IFN-g (2, 4, or 20 ng/ml) for 24 h.

Depletion of CD11c+ population with DT in lung ofCD11c-DTR Tg mice

PBS or DT (1 mg) was injected intranasally into CD11c-DTR Tg mice.After 24 h, recombinant murine IFN-g (3 mg) was administered intrana-sally into the mice. Total cells were collected from collagenase-treatedlung tissues or bronchoalveolar lavage fluid (BALF) 24 h after the ad-ministration of IFN-g into the PBS- or DT-treated CD11c-DTR Tg mice,and the number of cells was counted. CD11c- and CD11b-expressingpopulations were analyzed by flow cytometry, and the number ofCD11c+ cells was calculated. NKA levels in the collected BALF weremeasured by enzyme immunoassay 24 h after IFN-g administration.

Transduction of NK2R gene in bone marrow-derived DCs

Mouse NK2Rwith FLAG cDNAwas constructed by reverse transcription oftotal RNA from bone marrow-derived DCs with specific primers. Theresulting cDNA was inserted into pMX-IRES-GFP retroviral vector ob-tained from Dr. T. Kitamura (University of Tokyo). The transduction ofpMX-IRES-GFP (Mock) or pMX-NK2R-IRES-GFP into DCs was per-formed as described (36). For the following analyses, we sorted GFP+ cellsfrom the retrovirus-infected DCs using FACSAria. Briefly, the DCs wereharvested by pipetting and stained with anti-CD11c and 7AAD. The DCsgated by 7AAD2CD11c+GFP+ populations were sorted by FACSAria.Flow cytometry showed that the purity of the Mock- or NK2R-transducedDCs was .95% (data not shown). The NK2R-transduced DCs totallyexpressed NK2R-FLAG protein on the cell surface (data not shown).

Generation of human DCs from PBMCs

Research protocols for human subjects were approved by the InstitutionalReview Boards of Hokkaido University Graduate School of Medicine andthe Institute for Genetic Medicine. Written informed consent was obtainedfrom each subject. PBMCs were obtained from healthy individuals byFicoll-Hypaque (Amersham Bioscience, Uppsala, Sweden) gradient cen-trifugation after obtaining their informed consent. PBMCs (2 3 105 cells)were cultured with AIM-V (Invitrogen, Carlsbad, CA) in the presence ofrecombinant human IL-4 or IL-3 plus GM-CSF for 7 d, as describedpreviously (37). We evaluated phenotypes of the generated human DCs.We confirmed that .60% of whole-cell populations expressed CD11c andHLA class II, although .95% of adherent cells were CD11c+ and HLAclass II+ populations (data not shown). The generated human DCs were leftuntreated (control) or were treated with IFN-g (20 ng/ml) or poly I:C (20ng/ml) for 24 h, and the expression levels of human NK2R (hNK2R) orhuman TAC1 (hTAC1) were evaluated.

PCR

Total RNAwas extracted frommurine and human DCs with an Isogen RNAextraction kit (Nippon Gene, Toyama, Japan). First-strand cDNA wassynthesized using 1 mg total RNA with the oligo deoxythymidine primer(Invitrogen), at a 1:20 ratio, with Superscript III reverse transcriptase(Invitrogen). Genes for murine NK2R (Tarc2), IL-12p40 (Il12b), IFN-a(Ifna), IFN-b (Ifnb), b-actin (Actb), hNK2R (TACR2), hTAC1 (TAC1), andhuman GAPDH (GAPDH) were amplified using a thermal cycler (Light-Cycler; Roche, Indianapolis, IN). The primer sequences used in this studywere as follows: NK2R (forward: 59-AATGACAACGGAGGCAAGAT-39,reverse: 59-AAGCTGCAGGAATCACCACT-39, Universal probe: #4), IL-12p40 (forward: 59-GATTCAGACTCCAGGGGACA-39, reverse: 59-TGG-TTAGCTTCTGAGGACACATC-39, Universal probe: #27), IFN-a (for-ward: 59-CCTGTGTGATGCAGGAACC-39, reverse: 59-TCACCTCCCA-GGCACTGA-39, Probe: 59-AGACTCCCTGCTGGCTGTGAGGACA-39),IFN-b (forward: 59-ATGAGTGGTGGTTGCAGGC-39, reverse: 59-TGAC-CTTTCAAATGCAGTAGATTCA-39, Probe: 59-AAGCATCAGAGGCGG-ACTCTGGGA-39), TNF-a (forward: 59-GTTCTCTTCAAGGGACAAG-GCTG-39, reverse: 59-TCCTGGTATGAGATAGCAAATCGG-39, Probe:59-TACGTGCTCCTCACCCACACCGTCA-39), IL-6 (forward: 59-GCTA-

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CCAAACTGGATATAATCAGGA-39, reverse: 59-CCAGGTAGCTATGG-TACTCCAGAA-39, Probe: #6), b-actin (forward: 59-AGCCATGTACGT-AGCCATCCA-39, reverse: 59-TCTCCGGAGTCCATCACAATG-39, Probe:59-TGTCCCTGTATGCCTCTGGTCGTACCA-39), hNK2R (forward: 59-TCTACTCCATGACCGCCATT-39, reverse: 59-GGTGCTGGGAGCTGAA-AG-39, Universal probe: #11), hTAC1 (forward: 59-AGAATGTCACGTG-GGTCTGG-39, reverse: 59-CTCAGTGCCTTGCGGTATTT-39, Universalprobe: #75), and human GAPDH (forward: 59-CTCTGCTCCTCCTGT-TCGAC-39, reverse: 59-ACGACCAAATCCGTTGACTC-39, Universalprobe: #60). Sample signals were normalized to the housekeeping geneb-actin, according to the DDCt method: DCt = DCtsample 2 DCtreference.Percentages against the control sample were then calculated for eachsample. In some experiments, murine NK2R, IL-12p40, and b-actin wereamplified using a thermal cycler (PerkinElmer) with specific primers onthe basis of intron insertion. After amplification, PCR products with theappropriate size were separated by electrophoresis in 1% agarose gelscontaining ethidium bromide and visualized by UV light illumination.Primer sequences were as follows: NK2R (forward: 59-TGCTGGTGGC-TGTAACAGGCAACG-39, reverse: 59-TAGAAACATTGTGGGGAGGC-GAGAGC-39), IL-12p40 (forward: 59-TCCTGAAGTGTGAAGCACCA-AA-39, reverse: 59-GAGTGCTCCAGGAGTCAGGGTA-39), and b-actin(forward: 59-GTGATGGTGGGAATGGGTCAG-39, reverse: 59-TTTGAT-GTCACGCACGATTTCC-39).

Flow cytometry

For analysis of cell surface molecules, the DCs were stained with 7AADand fluorescence-labeled anti-CD11b, anti-CD11c, and anti-NK2R mAbs.Expression levels were evaluated by FACSCalibur or FACSCanto (BDBiosciences).

Confocal microscopy

DCs generated on glass coverslips in 35-mm culture dishes (Iwaki) werestimulated with IFN-g for 24 h and fixed with 4% paraformaldehyde inPBS. Immunostaining of DCs was performed with permeabilization by BDPerm/Wash Buffer containing Abs and 1% goat serum. The cells werewashed with PBS and mounted with Mounting Medium (DakoCytoma-tion). We used the anti-NK2R mAb at 1:50 dilution. Hoechst 33352 wasused for nuclear staining. Fluorescence signals were detected with thespectral Confocal Scanning system (FV300; Olympus, Tokyo, Japan)through a 633/1.4 Oil DIC I lens. The signals were visualized with thestandard software (Olympus).

T cell-activation assay

Mock- or NK2R-overexpressing DCs were used as APCs. OVA peptide-specific CD4+ and CD8+ T cells were prepared from OT-II and OT-ITCR-Tg mice using a nylon wool column and an IMag Cell SeparationSystem with anti-CD4 or anti-CD8 mAb-bound beads, respectively. Thepurities of CD4+ or CD8+ T cells were .95% (data not shown). Mock- orNK2R-DCs (2 3 104 cells) were cultured with OT-II CD4+ T cells (1 3105 cells) in the presence of vehicle or NKA (1 mM) with OT-II peptide (0,0.25, 1 mg/ml) for 48 h. [3H]thymidine was added to the culture mediaduring the last 4 h. [3H]thymidine incorporation by the proliferated cellswas counted using MicroBeta2 (PerkinElmer). IL-2 production wasassayed with Immunoassay kits (BD Bioscience), as described (35). Inanother experiment, normal DCs from bone marrow of wild-type micewere sorted and used as APCs. OT-II or OT-I TCR-Tg CD4+ or CD8+

T cells were prepared by the same methods described above. Normal DCswere cultured together with OT-II CD4+ T cells or OT-I CD8+ T cells in the

FIGURE 1. IFN-g significantly upregulates NK2R

mRNA level of DCs in a STAT1-dependent manner.

(A) Bone marrow-derived DCs were generated from

wild-type mice. Quantitative PCR analysis was used to

evaluate NK2R mRNA expression level in the DCs

treated with IFN-g (10 ng/ml) for 6 h. Mean and SD

were calculated from data of three independent

experiments (n = 6/group). (B) NK2R mRNA expres-

sion levels in wild-type and Stat12/2 DCs treated with

IFN-g (10 ng/ml) for 6 h were evaluated by PCR. One

representative of at least three independent experi-

ments is shown. Similar results were obtained in the

experiments. (C) NK2R protein expression of untreated

DCs (left panel; control) or DCs treated with 10 ng/ml

IFN-g for 24 h (right panel) was evaluated by confocal

microscopy. One representative of at least three inde-

pendent experiments is shown. Scale bars, 5 mm. (D)

Surface NK2R expression levels of the control and

IFN-g–treated DCs were analyzed by flow cytometry.

One representative of at least three independent

experiments is shown. (E) Mean fluorescence intensity

(MFI; left panel) and percentage (right panel) of

NK2R-expressing cells in the control and IFN-g–

treated DCs. Mean and SD were calculated from data

of three independent experiments (n = 6/group). *p ,0.05, Student t test.

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presence of NK2R antagonist (GR: 10 mM) and OT-II peptide (0, 0.25, 1mg/ml), OT-I peptide (0, 0.25, 1 mg/ml) for 24–72 h, or OVA protein (0, 25,100 mg/ml) for 72 h, respectively. IL-2, IFN-g, IL-4, and IL-17 productionwas evaluated with the Immunoassay kits.

ELISA

IL-2, IFN-g, IL-4, IL-17, TNF-a, and IL-6 levels in the culture super-natants were measured using OptEIA mouse IL-2, IFN-g, IL-4, IL-17,TNF-a, and IL-6 ELISA kits, respectively (BD Biosciences). IFN-a andIFN-b levels in the culture supernatants were measured with VeriKineMouse IFN-a and IFN-b ELISA kits (PBL InterferonSource, Piscataway,NJ). ELISA kits were used according to the manufacturer’s instructions.The NKA level was determined using a Neurokinin A Enzyme Immuno-assay kit (Peninsula Laboratories, San Carlos, CA).

Statistical analysis

All experiments were independently repeated at least three times. Mean andSD were calculated for in vitro murine data, and mean and SE were cal-culated for in vivomurine and in vitro human data. Significant differences inthe results were determined by the two-tailed Student t test; p , 0.05 wasconsidered statistically significant.

ResultsIFN-g stimulation enhances NK2R expression level of DCs

To address the effect of neuropeptide signaling on DC-mediatedimmune responses, we first investigated the expression level ofa receptor for NKA, NK2R, on DCs in a type 1 immune condition.DCs generated from bone marrow cells of wild-type mice werestimulated with recombinant murine IFN-g in vitro. As a result,IFN-g treatment significantly induced NK2R mRNA expressionon DCs (Fig. 1A, 1B). The NK2R mRNA induction by IFN-gstimulation was remarkably reduced in STAT-1–deficient DCs(Fig. 1B). These data indicate that IFN-g stimulation inducesNK2R mRNA expression of DCs in a STAT-1–dependent manner.In addition, we found that LPS stimulation induced NK2R mRNAexpression on DCs (Supplemental Fig. 1A). The LPS-dependentaugmentation of NK2R mRNA level was not observed inIFNAR1- or STAT-1–deficient DCs (Supplemental Fig. 1A, 1B).These data suggest that type 1 IFN produces after LPS stimula-tion induced NK2R mRNA expression of DCs by an autocrinemechanism in a STAT-1–dependent manner.We further evaluated whether IFN-g enhanced NK2R protein

expression levels of DCs. Confocal microscopy showed thatNK2R protein level was enhanced in the IFN-g–stimulated DCs(Fig. 1C). We confirmed that both the surface expression levels ofNK2R on DCs (Fig. 1D) and the percentage of NK2R-expressingcells were significantly enhanced by IFN-g stimulation (Fig. 1E).These data suggest that IFN-g, type 1 cytokine stimulation acti-vates the NK2R-mediated signaling cascade of DCs in a STAT-1–dependent manner.

DCs elevate the NKA, a neuropeptide, level in response toIFN-g stimulation in vivo and in vitro

In the current study, we found that intranasal injection of IFN-ginto wild-type mice significantly enhanced the NK2R mRNAexpression level of lung tissue and the NKA level in BALF,resulting in increased airway inflammation (11). In the currentstudy, we investigated whether DCs were required for the increasein NKA. CD11c-DTR Tg mice were intranasally injected withPBS as a control or DT and then IFN-g was injected into the mice.We confirmed that CD11c+ populations in the lung tissue andBALF were remarkably reduced by DT treatment (Fig. 2A). At 24h after the IFN-g injection, NKA level in BALF was significantlyreduced by the depletion of the CD11c+ population (Fig. 2B).These findings suggest that CD11c+ populations, such as pulmo-nary DCs and alveolar macrophages, contribute to IFN-g–depen-dent NKA elevation in the lung. Furthermore, we determined that

NKA level in the culture supernatant of bone marrow-derived DCstreated with IFN-g. As a result, the NKA level increased signifi-cantly at 24 h after the IFN-g stimulation (Fig. 2C). Taken to-gether, these findings indicate that DCs are at least one of theresponsible populations for the increase in NKA levels in responseto IFN-g stimulation.

NKA stimulation significantly induces IFN-a/b mRNAexpression levels on DCs

We further investigated the effect of NKA on the production ofcytokines by DCs. NKA stimulation significantly enhanced mRNAexpression levels of both IFN-a (Fig. 3A) and IFN-b (Fig. 3B),whereas IL-12p40 increased only slightly (Fig. 3C), and TNF-a(Fig. 3D) and IL-6 (Fig. 3E) were unchanged. In this study, weconfirmed that LPS, a TLR4 ligand, stimulation significantly en-hanced IFN-b, IL-12p40, TNF-a, and IL-6 but not IFN-a. Thesefindings suggest that NKA stimulation induces type 1 IFN mRNAsby DCs. Furthermore, we evaluated IFN-a, IFN-b, IL-12p70,TNF-a, and IL-6 protein production by NKA-stimulated DCs.However, we could not detect these protein production levels afterthe NKA stimulation in the present experiments (data not shown).

Transduction of NK2R gene into DCs enhances Ag-specificIL-2 production by CD4+ T cells

To evaluate the effects of NK2R-mediated NKA stimulation on DCfunctions, we constructed the pMX-FLAG-NK2R-IRES-GFP ret-

FIGURE 2. DCs are required for IFN-g–dependent NKA increase. IFN-g

(3 mg) was intranasally administered into CD11c-DTRTg mice preinjected

with PBS (n = 7/group) or DT (n = 6/group). (A) The percentage and total

number of CD11c+ cells in lung tissue (left panels) and BALF (right

panels) were analyzed by flow cytometry at 24 h after IFN-g adminis-

tration. Mean and SE were calculated from data of three independent

experiments (n = 6–7/group). (B) NKA levels in the collected BALF at 24 h

after IFN-g treatment in vivo. Mean and SE were calculated from data of

three independent experiments (n = 6–7/group). (C) NKA levels in the

culture supernatants of bone marrow-derived DCs treated with IFN-g (10

ng/ml) for 24 h. Mean and SD were calculated from data of three inde-

pendent experiments (n = 6–7/group). *p , 0.05, Student t test.

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roviral vector. We then confirmed the surface expression levelof FLAG-tagged NK2R protein in Plat E cells by flow cytometry(data not shown) and alteration of the morphology of NK2R-overexpressed RAW264.7 cells compared with Mock-RAW264.7cells after NKA stimulation (Supplemental Fig. 2A).We transduced NK2R gene into DCs with a retrovirus-infection

system, and the Mock- or NK2R-transduced DCs were isolatedusing a cell sorter. We found that the NK2R-DCs enhanced surfaceMHC class II expression level after NKA stimulation comparedwith Mock-DCs (Fig. 4A), although IL-12 production by Mock- orNK2R-DCs was not observed by flow cytometry (SupplementalFig. 2C). Then, we cocultured OT-II CD4+ T cells with Mock- orNK2R-transduced DCs in the presence or absence of OT-II pep-tides plus NKA or vehicle. The transduction of NK2R gene intoDCs appeared to promote the Ag-specific responses to CD4+

T cells (Fig. 4B). Then, we evaluated the proliferation by [3H]thy-midine-incorporation assay and confirmed that NK2R-DCs sig-nificantly enhanced cell proliferation compared with Mock-DCs.In addition, NKA stimulation increased the enhancement of cellproliferation (Fig. 4C). Furthermore, we examined IL-2 produc-tion by CD4+ T cells cocultured with Mock- or NK2R-transducedDCs in the presence of NKA with or without OT-II peptide.NK2R-DCs significantly enhanced Ag-specific IL-2 production byCD4+ T cells compared with Mock-DCs. NKA stimulation aug-mented IL-2 production by CD4+ T cells cocultured with NK2R-DCs (Fig. 4D). These data suggest that NK2R-dependent NKAstimulation increases Ag-presentation of DCs to activate CD4+

T cells.In the present experiments, the proliferation and IL-2 produc-

tion by CD4+ T cells were significantly increased by the NK2R

transduction into DCs, even in the absence of extraneous NKAstimulation. We found that the culture medium with 10% FCScontained NKA, as shown in the normal culture condition (Fig.2C), suggesting that the NKA in the culture medium also affectedMHC class II expression of NK2R-transduced DCs without thefurther addition of NKA.

Blockade of NK2R-mediated signaling pathway significantlyinhibits Ag-specific CD4+ and CD8+ T cell responses

To confirm the effect of NKA–NK2R signaling on Ag-specificT cell responses, we cultured spleen cells obtained from OT-IIor OT-I mice in the presence of NK2R antagonist GR for 48 h.Immune responses to each OVA Ag peptide were evaluated by[3H]thymidine-incorporation assay. The Ag-specific proliferationof spleen cells from OT-II and OT-I mice was significantly sup-pressed by the addition of GR (10 mM; Supplemental Fig. 3A).We confirmed that there was no difference in the ratio of 7AAD+

Annexin V+ apoptotic cells between GR and DMSO controlgroups at 8 h (Supplemental Fig. 3B).Then, we cocultured OT-II CD4+ T cells with DCs in the

presence of GR (10 mM). The blockade of NK2R-mediated sig-naling by antagonist remarkably reduces IFN-g and IL-2 pro-duction by CD4+ T cells in the presence of OT-II peptide or OVAprotein (Fig. 5A). Under the same conditions, neither IL-17 norIL-4 production was observed in the present coculture system(data not shown). Furthermore, we examined Ag-specific re-sponses of OT-I CD8+ T cells cocultured with DCs in the presenceof GR. The blockade of NK2R-mediated signaling also suppressesIFN-g production by CD8+ T cells after stimulation with OT-Ipeptide or OVA protein (Fig. 5B).

FIGURE 3. NKA stimulation enhances both IFN-

a/b and NK2R mRNA expression levels of DCs. DCs

generated from wild-type mice were treated with NKA

(1 mM) or LPS (1 mg/ml) for 6 h. The mRNA ex-

pression levels of IFN-a (A), IFN-b (B), IL-12p40 (C),

TNF-a (D), and IL-6 (E) were determined by quanti-

tative PCR. Mean and SD were calculated from data of

three independent experiments (n = 6/group). *p ,0.05, Student t test.

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Thus, these findings strongly suggest that the NKA–NK2R-signaling cascade is involved in DC-mediated type 1 immunityincluding Ag-specific CD4+ and CD8+ T cell responses.

Human DCs induce NK2R and TAC1 genes after IFN-g andpoly I:C stimulation

Finally, we examined whether human DCs induce expressionof NK2R (hNK2R) and TAC1 (hTAC1) after IFN-g stimulation.CD11c-, HLA class II-, and HLA class I-expressing DCs weregenerated from adherent cells of human PBMCs and stimulatedwith recombinant human IFN-g for 24 h. As a result, we con-firmed that both hNK2R protein level (Fig. 6A) and surface ex-pression levels (Fig. 6B, 6C) were remarkably enhanced by IFN-gstimulation, similar to the case observed in mouse DCs. In thisstudy, we found that both hNK2R and hTAC1 mRNA expression

levels were significantly enhanced by IFN-g stimulation (Fig. 6D,6E). Furthermore, human DCs were stimulated with poly I:C,dsRNA, and IFN-g for 24 h. We found that poly I:C, a TLR3 li-gand, enhanced both hNK2R and hTAC1 mRNA expression levelsin human DCs (Fig. 6D, 6E). In this study, we confirmed thathuman DCs enhanced HLA class II expression levels after NKAplus IFN-g stimulation compared with IFN-g alone, whereas NKAstimulation did not induce cytokine production (data not shown).These findings suggest that NKA–NK2R signaling could regulateDC function in human immunity.

DiscussionExcitatory transmitters, such as tachykinins including SP andNKA, are widely distributed within both the central and the pe-ripheral nervous systems. Although the tachykinins were initiallyconsidered neurotransmitters, many articles reported that expres-sion of their receptors, NK1R and NK2R, was observed in non-neural tissues, including endothelial cells, fibroblasts, smooth

FIGURE 4. NK2R-mediated NKA signaling in DCs promotes the acti-

vation of Ag-specific CD4+ T cells. DCs generated from wild-type mice

were transduced with Mock or NK2R genes with the retroviral infection

system using pMX-IRES-GFP or pMX-NK2R-IRES-GFP, respectively.

(A) Surface MHC class II (I-Ab) expression levels of Mock- and NK2R-

transduced DCs treated with vehicle or NKA for 24 h were evaluated by

flow cytometry. Data are from one representative experiment of at least

three independent experiments. Similar results were obtained in the

experiments. (B) OT-II CD4+ T cells (1 3 105 cells) were cocultured with

Mock- or NK2R-transduced DCs (2 3 104 cells) in the presence of vehicle

or NKA (1 mM) with OT-II peptide (1 mg/ml). Photomicrographs of the

culture cells at 48 h after stimulation. Data are from one representative

experiment of at least three independent experiments. Scale bars, 250 mm.

(C) Proliferation of OT-II CD4+ T cells cocultured with NK2R- or Mock-

transduced DCs in the presence of vehicle or NKA (1 mM) with OT-II

peptide (0, 0.25, 1 mg/ml) were evaluated by [3H]thymidine-incorporation

assay. Mean and SD were calculated from the representative data of three

independent experiments (n = 3/group). (D) IL-2 production levels of

OT-II CD4+ T cells cocultured with Mock- or NK2R-transduced DCs in

the presence of vehicle or NKA (1 mM) with OT-II peptide were evaluated

by ELISA. Mean and SD were calculated from data of three independent

experiments (n = 3/group). *p , 0.05, Student t test.

FIGURE 5. Blockade of NK2R-mediated signaling cascade suppresses

IFN-g and IL-2 production by Ag-specific T cells. DCs were generated

from bone marrow cells of wild-type mice in the presence of GM-CSF for

6 d. (A) The generated DCs (2 3 104 cells) were cocultured with OT-II

CD4+ T cells (1 3 105 cells) in the absence or presence of NK2R an-

tagonist (GR, 10 mM) with OT-II peptide (right panels) or OVA protein

(left panels) for 24–72 h. IFN-g production by the OT-II CD4+ T cells at 72 h

(top panels) and IL-2 production at 24 h (bottom panels) were evaluated by

ELISA. Mean and SD were calculated from data of three independent

experiments (n = 6/group). (B) The generated DCs (2 3 104 cells) were

cocultured with OT-I CD8+ T cells (1 3 105 cells) in the absence or

presence of NK2R antagonist (GR, 10 mM) with OT-I peptide (right panel)

or OVA protein (left panel) for 72 h. IFN-g production by the OT-I CD8+

T cells were evaluated by ELISA. Mean and SD were calculated from data

of three independent experiments (n = 6/group). *p , 0.05, Student t test.

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muscle cells, and inflammatory cells, suggesting that tachykininsfunction between the nervous system and other organs (27–33).In this study, we found that both murine and human DCs, one of

the most powerful APCs, significantly enhanced NKA productionlevels and expressed NK2R after IFN-g stimulation (Figs. 1, 6). Inaddition, stimulation of DCs with TLR ligands, such as LPS andpoly I:C, which mimic bacterial and virus infection, respectively,enhanced NK2R expression levels (Fig. 6, Supplemental Fig. 1).In addition, we revealed in this study that murine DCs stimulatedwith NKA significantly induced mRNA expression of IFN-a andIFN-b, type 1 IFNs (Fig. 3). Although the protein expressions ofthese cytokines were not detected in the present experiments, weconfirmed the blocking effects of anti–IFN-b and anti–IL-12mAbs on NKA-dependent augmentation of IFN-g production af-ter OVA Ag stimulation (data not shown). Therefore, we specu-lated that NKA-mediated cytokine production might function inthe microenvironment, including cell-to-cell interaction. Takentogether, these findings strongly suggest that NK2R-mediatedneuropeptide signaling is involved in host immune responses,including cytokine production and Ag presentation by DCs.Previous works indicated that blockade of NK1R influences host

responses against various infections. In salmonellosis, mice treatedwith an SP antagonist have increased bacterial burden and reducedIFN-g and IL-12 production compared with appropriate controls

(38, 39). Mice lacking NK1R reduced schistosomiasis granulomaformation (40). In a posttreatment-reactive encephalopathy modelof Trypanosoma brucei, NK1R-deficient mice have a significantlyreduced clinical impairment, but a more severe neuroinflammatoryresponse, compared with wild-type mice (41). The critical roles ofSP were reported in T cell-mediated experimental autoimmuneencephalomyelitis models (42, 43). The findings strongly suggestthat the SP–NK1R–signaling cascade contributes to type 1 im-munity, as well as inflammatory responses. Although NK1R isubiquitously expressed in addition to the CNS, it was demon-strated that NK2R is mostly expressed in peripheral tissues (29–33). Although the precise function of the NKA–NK2R–signalingpathway, especially in immune responses, is less-well knowncompared with that of NK1R, we demonstrated in this study thatNK2R-mediated NKA stimulation enhances DC function, suchas IFN-a/b production (Fig. 3A, 3B), MHC class II expression(Fig. 4A), and Ag presentation to CD4+ and CD8+ T cells (Figs.4B, 4C, 5).NK2R, as well as NK1R, belongs to the rhodopsin-like family 1

of G protein-coupled receptors, which plays a crucial role in in-tracellular signaling (27, 28). Previously, we indicated that theTRIF–GEFH1–RhoB pathway regulated MHC class II expressionon DCs, which was required for the subsequent CD4+ T cell ac-tivation (44). In this study, we indicated that the surface MHC

FIGURE 6. Human DCs induce both NK2R and

TAC1 gene after IFN-g or poly I:C stimulation. Hu-

man DCs generated from the adherent cells of PBMCs

were left untreated (control) or were treated with

IFN-g (20 ng/ml) or poly I:C (20 ng/ml) for 24 h. (A)

hNK2R protein expression levels of the control (left

panel) and IFN-g–treated (right panel) human DCs

were evaluated by confocal microscopy. One repre-

sentative experiment of at least three independent ex-

periments is shown. Similar results were obtained in

the experiments. Scale bars, 5 mm. (B) Surface hNK2R

expression levels of the control and IFN-g–treated

human DCs were determined by flow cytometry. One

representative experiment of at least three independent

experiments is shown. (C) Mean fluorescence intensity

(MFI; left panel) and percentage (right panel) of

hNK2R-expressing cells in the control and IFN-g–

treated human DCs were evaluated by flow cytometry.

Mean and SE were calculated from data of three in-

dependent experiments (n = 6/group). (D) Relative

expression levels of hNK2R and TAC1 mRNAs in the

human DCs treated with IFN-g, poly I:C, or NKA for

24 h were determined by quantitative PCR. Mean and

SE were calculated from data of three independent

experiments (n = 6/group). (E) hTAC1 mRNAs in the

human DCs treated with IFN-g or poly I:C for 24 h

were determined by quantitative PCR. Mean and SE

were calculated from data of three independent ex-

periments (n = 6/group). *p , 0.05, Student t test.

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class II expression level of NK2R-transduced DCs was increasedby NKA stimulation (Fig. 4A). Moreover, the morphology ofNK2R-transduced DCs was altered by the addition of NKA (Sup-plemental Fig. 2B) in addition to NK2R-transduced RAW264.7cells (Supplemental Fig. 2A). These findings suggest that NK2R-mediated signaling in DCs might be related to membrane traf-ficking of MHC class II vesicles and alteration of cytoskeletonmediated by a posttranscriptional mechanism.NKA stimulation significantly enhanced mRNA expression

levels of type 1 IFNs of DCs rather than IL-12p40, although it didnot induce mRNA expression levels of TNF-a and IL-6 (Fig. 3,Supplemental Fig. 2C). In general, plasmacytoid DCs induce bothIFN-a and IFN-b production in response to foreign nucleic acidsthrough TLR7 and TLR9 (45). In contrast, conventional DCs andmacrophages produce IFN-b and various proinflammatory cyto-kines by TLR3, TLR7, and TLR9 ligands after stimulation (46).Although DCs generated from bone marrow with GM-CSF in thisstudy showed phenotypes of conventional DCs, IFN-a and IFN-bmRNA expression was induced by NKA stimulation (Fig. 3A,3B). The present data suggest that NK2R-dependent neuropeptidesignaling uses a different mechanism than the TLR-dependentpathway for cytokine production by conventional DCs.Recently, we demonstrated that intranasal administration of

IFN-g remarkably induced NKA and NK2R expression in the lungtissues and blockade of NKA–NK2R signaling significantly sup-pressed IFN-g–induced AHR and the symptom of Th1 cell-transferred asthma model (11). These findings suggested that theNKA–NK2R cascade must be related to neuro-immune cross-talkin type 1 immunity including airway inflammation. We found inthis study that depletion of CD11c+ cells from lung tissues re-markably reduced the production level of NKA after intranasalIFN-g injection in vivo (Fig. 2B). We also confirmed that NK2Rexpression was induced in alveolar macrophages collected fromBALF, in addition to macrophages from peritoneal exudate cellsand spleen CD11c+ DCs after IFN-g stimulation (data not shown).Thus, we speculated that both pulmonary DCs and alveolarmacrophages might regulate type 1 immunity through the NK2R-mediated signaling pathway in the lung. Then, we focused on theprecise effect of NKA–NK2R signaling on the immunologicalfunction of DCs. NK2R-transduced DCs increased MHC class IIlevels, resulting in the enhanced proliferation of CD4+ T cells andIL-2 production by CD4+ T cells after the Ag stimulation (Fig. 4).In addition, NK2R selective antagonist strongly suppressed bothIFN-g and IL-2 production by CD4+ T cells and IFN-g productionby CD8+ T cells cocultured with Ag-loaded DCs (Fig. 5). IL-4and IL-17 production by CD4+ T cells did not increase in theseexperiments (data not shown). Thus, NK2R-dependent neuro-peptide signaling would be involved in host inflammatoryresponses induced by type 1 immunity via the augmentation ofDC functions (Supplemental Fig. 4).Chronic inflammatory pulmonary disorders, such as various

infections, and severe asthma, including childhood asthma andCOPD of smokers, have become a major public health problemworldwide. Although the pathology of severe asthma occasionallyshows steroid drug resistance, the precise mechanisms remainunknown compared with Th2-mediated bronchial asthma. Weestablished Th1-, Th2-, and Th17-dependent asthma models byadoptive transfer of these effector cells into wild-type mice (9, 10)and confirmed that the Th1 and Th17 models were steroid resis-tant, whereas the Th2-mediated asthma model was steroid sensi-tive. Then, we confirmed that administration of NK2R antagonistsignificantly blocked the Th1 cell-dependent AHR elevation.Therefore, we have a high expectation that blockade of the NKA–NK2R–signaling pathway would be a powerful therapy for pa-

tients with steroid-resistance disorders, including severe inflam-matory diseases.In the present work, we found that human DCs also induced

expression of hNKA and hTAC1 mRNAs in response to IFN-g(Fig. 6). Furthermore, stimulation of the TLR3 ligand poly I:Cenhanced both genes. Although human CD11c+ and HLA class II+

cells were possible contaminants of both monocyte and macro-phages in the present experiments, these findings indicate thatNK2R-dependent cross-talk between the neural and immunesystems would function in human subjects. Therefore, we are nowevaluating the effect of NK2R antagonist on the activation ofhuman immune cells and confirming the several suppressioneffects of cytokine production by human DCs and T cells.We conclude that NK2R-dependent neuropeptide signaling

activates DC-mediated immune responses, suggesting that theNKA/NK2R-mediated signaling pathway would be a promisingtarget in therapy for patients with chronic inflammation caused byexcessive activation of type 1 immunity, including steroid-resistantsevere asthma.

AcknowledgmentsWe thank Dr. T. Kitamura (University of Tokyo) for providing pMX vector

plasmid and Dr. T. Sudo (Toray, Tokyo, Japan) for providing GM-CSF–

producing CHO cells.

DisclosuresThe authors have no financial conflicts of interest.

References1. Lloyd, C. M., and E. M. Hessel. 2010. Functions of T cells in asthma: more than

just T(H)2 cells. Nat. Rev. Immunol. 10: 838–848.2. Barrett, N. A., and K. F. Austen. 2009. Innate cells and T helper 2 cell immunity

in airway inflammation. Immunity 31: 425–437.3. Kim, H. Y., R. H. DeKruyff, and D. T. Umetsu. 2010. The many paths to asthma:

phenotype shaped by innate and adaptive immunity. Nat. Immunol. 11: 577–584.4. Umetsu, D. T., and R. H. Dekruyff. 2010. Natural killer T cells are important in

the pathogenesis of asthma: the many pathways to asthma. J. Allergy Clin.Immunol. 125: 975–979.

5. Bush, A., and S. Saglani. 2010. Management of severe asthma in children.Lancet 376: 814–825.

6. Ohnishi, H., N. Miyahara, and E.W. Gelfand. 2008. The role of leukotriene B(4)in allergic diseases. Allergol. Int. 57: 291–298.

7. Barnes, P. J. 2008. Immunology of asthma and chronic obstructive pulmonarydisease. Nat. Rev. Immunol. 8: 183–192.

8. Barnes, P. J. 2008. The cytokine network in asthma and chronic obstructivepulmonary disease. J. Clin. Invest. 118: 3546–3556.

9. Ashino, S., D. Wakita, Y. Shiohama, Y. Iwakura, K. Chamoto, T. Ohkuri,H. Kitamura, and T. Nishimura. 2010. A T(h)17-polarized cell population thathas infiltrated the lung requires cells that convert to IFN-gamma production inorder to induce airway hyperresponsiveness. Int. Immunol. 22: 503–513.

10. Ashino, S., D. Wakita, Y. Zhang, K. Chamoto, H. Kitamura, and T. Nishimura.2008. CpG-ODN inhibits airway inflammation at effector phase through down-regulation of antigen-specific Th2-cell migration into lung. Int. Immunol. 20:259–266.

11. Kobayashi, M., S. Ashino, Y. Shiohama, D. Wakita, H. Kitamura, andT. Nishimura. 2012. IFN-g elevates airway hyper-responsiveness via up-regulation of neurokinin A/neurokinin-2 receptor signaling in a severe asthmamodel. Eur. J. Immunol. 42: 393–402.

12. Groneberg, D. A., S. Harrison, Q. T. Dinh, P. Geppetti, and A. Fischer. 2006.Tachykinins in the respiratory tract. Curr. Drug Targets 7: 1005–1010.

13. De Swert, K. O., and G. F. Joos. 2006. Extending the understanding of sensoryneuropeptides. Eur. J. Pharmacol. 533: 171–181.

14. Patterson, R. N., B. T. Johnston, J. E. Ardill, L. G. Heaney, and L. P. McGarvey.2007. Increased tachykinin levels in induced sputum from asthmatic and coughpatients with acid reflux. Thorax 62: 491–495.

15. Mostafa, G. A., S. M. Reda, M. M. Abd El-Aziz, and S. A. Ahmed. 2008.Sputum neurokinin A in Egyptian asthmatic children and adolescents: relation toexacerbation severity. Allergy 63: 1244–1247.

16. Schelfhout, V., V. Van De Velde, C. Maggi, R. Pauwels, and G. Joos. 2009. Theeffect of the tachykinin NK(2) receptor antagonist MEN11420 (nepadutant) onneurokinin A-induced bronchoconstriction in asthmatics. Ther. Adv. Respir. Dis.3: 219–226.

17. Quartara, L., M. Altamura, S. Evangelista, and C. A. Maggi. 2009. Tachykininreceptor antagonists in clinical trials. Expert Opin. Investig. Drugs 18: 1843–1864.

8 NEUROKININ A SIGNALING CONTROLS DC-MEDIATED T CELL RESPONSES

by guest on March 31, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

18. Boot, J. D., S. de Haas, S. Tarasevych, C. Roy, L. Wang, D. Amin, J. Cohen,P. J. Sterk, B. Miller, A. Paccaly, et al. 2007. Effect of an NK1/NK2 receptorantagonist on airway responses and inflammation to allergen in asthma. Am. J.Respir. Crit. Care Med. 175: 450–457.

19. Theoharides, T. C., B. Zhang, D. Kempuraj, M. Tagen, M. Vasiadi,A. Angelidou, K. D. Alysandratos, D. Kalogeromitros, S. Asadi, N. Stavrianeas,et al. 2010. IL-33 augments substance P-induced VEGF secretion from humanmast cells and is increased in psoriatic skin. Proc. Natl. Acad. Sci. USA 107:4448–4453.

20. Monaco-Shawver, L., L. Schwartz, F. Tuluc, C. J. Guo, J. P. Lai, S. M. Gunnam,L. E. Kilpatrick, P. P. Banerjee, S. D. Douglas, and J. S. Orange. 2011. SubstanceP inhibits natural killer cell cytotoxicity through the neurokinin-1 receptor. J.Leukoc. Biol. 89: 113–125.

21. Barros, P. O., T. B. Ferreira, M. M. Vieira, C. R. Almeida, C. F. Araujo-Lima,R. G. Silva-Filho, J. Hygino, R. M. Andrade, A. F. Andrade, and C. A. Bento.2011. Substance P enhances Th17 phenotype in individuals with generalizedanxiety disorder: an event resistant to glucocorticoid inhibition. J. Clin. Immu-nol. 31: 51–59.

22. Blum, A., T. Setiawan, L. Hang, K. Stoyanoff, and J. V. Weinstock. 2008.Interleukin-12 (IL-12) and IL-23 induction of substance p synthesis in murineT cells and macrophages is subject to IL-10 and transforming growth factor betaregulation. Infect. Immun. 76: 3651–3656.

23. Sun, J., R. D. Ramnath, R. Tamizhselvi, and M. Bhatia. 2009. Role of proteinkinase C and phosphoinositide 3-kinase-Akt in substance P-induced proin-flammatory pathways in mouse macrophages. FASEB J. 23: 997–1010.

24. Chernova, I., J. P. Lai, H. Li, L. Schwartz, F. Tuluc, H. M. Korchak, S. D. Douglas,and L. E. Kilpatrick. 2009. Substance P (SP) enhances CCL5-induced chemotaxisand intracellular signaling in human monocytes, which express the truncatedneurokinin-1 receptor (NK1R). J. Leukoc. Biol. 85: 154–164.

25. Pavlovic, S., C. Liezmann, S. M. Blois, R. Joachim, J. Kruse, N. Romani,B. F. Klapp, and E. M. Peters. 2011. Substance P is a key mediator of stress-induced protection from allergic sensitization via modified antigen presentation.J. Immunol. 186: 848–855.

26. Gleason, N. R., G. Gallos, Y. Zhang, and C. W. Emala. 2010. Propofol prefer-entially relaxes neurokinin receptor-2-induced airway smooth muscle contrac-tion in guinea pig trachea. Anesthesiology 112: 1335–1344.

27. Tuluc, F., J. P. Lai, L. E. Kilpatrick, D. L. Evans, and S. D. Douglas. 2009.Neurokinin 1 receptor isoforms and the control of innate immunity. TrendsImmunol. 30: 271–276.

28. Douglas, S. D., and S. E. Leeman. 2011. Neurokinin-1 receptor: functionalsignificance in the immune system in reference to selected infections and in-flammation. Ann. N. Y. Acad. Sci. 1217: 83–95.

29. Sculptoreanu, A., F. Aura Kullmann, and W. C. de Groat. 2008. Neurokinin 2receptor-mediated activation of protein kinase C modulates capsaicin responsesin DRG neurons from adult rats. Eur. J. Neurosci. 27: 3171–3181.

30. Sun, J., R. D. Ramnath, R. Tamizhselvi, and M. Bhatia. 2008. Neurokinin Aengages neurokinin-1 receptor to induce NF-kappaB-dependent gene expressionin murine macrophages: implications of ERK1/2 and PI 3-kinase/Akt pathways.Am. J. Physiol. Cell Physiol. 295: C679–C691.

31. Miotto, D., P. Boschetto, G. Cavallesco, E. Zeni, P. Querzoli, M. Pedriali,S. Chiarelli, L. M. Fabbri, and C. E. Mapp. 2007. Increased neurokinin-2 re-ceptor expression in alveolar macrophages of smokers with COPD. Histopa-thology 51: 128–131.

32. Michalski, C. W., X. Shi, C. Reiser, P. Fachinger, A. Zimmermann,M. W. Buchler, P. Di Sebastiano, and H. Friess. 2007. Neurokinin-2 receptorlevels correlate with intensity, frequency, and duration of pain in chronic pan-creatitis. Ann. Surg. 246: 786–793.

33. Hernandez, J. M., G. Cox, and L. J. Janssen. 2008. Involvement of theneurokinin-2 receptor in airway smooth muscle stretch-activated contractionsassessed in perfused intact bovine bronchial segments. J. Pharmacol. Exp. Ther.327: 503–510.

34. Beresford, I. J., R. L. Sheldrick, D. I. Ball, M. P. Turpin, D. M. Walsh,A. B. Hawcock, R. A. Coleman, R. M. Hagan, and M. B. Tyers. 1995.GR159897, a potent non-peptide antagonist at tachykinin NK2 receptors. Eur. J.Pharmacol. 272: 241–248.

35. Kitamura, H., H. Morikawa, H. Kamon, M. Iguchi, S. Hojyo, T. Fukada,S. Yamashita, T. Kaisho, S. Akira, M. Murakami, and T. Hirano. 2006. Toll-likereceptor-mediated regulation of zinc homeostasis influences dendritic cellfunction. Nat. Immunol. 7: 971–977.

36. Kitamura, H., H. Kamon, S. Sawa, S. J. Park, N. Katunuma, K. Ishihara,M. Murakami, and T. Hirano. 2005. IL-6-STAT3 controls intracellular MHCclass II alphabeta dimer level through cathepsin S activity in dendritic cells.Immunity 23: 491–502.

37. Ohkuri, T., D. Wakita, K. Chamoto, Y. Togashi, H. Kitamura, and T. Nishimura.2009. Identification of novel helper epitopes of MAGE-A4 tumour antigen:useful tool for the propagation of Th1 cells. Br. J. Cancer 100: 1135–1143.

38. Kincy-Cain, T., and K. L. Bost. 1996. Increased susceptibility of mice to Sal-monella infection following in vivo treatment with the substance P antagonist,spantide II. J. Immunol. 157: 255–264.

39. Kincy-Cain, T., J. D. Clements, and K. L. Bost. 1996. Endogenous and exoge-nous interleukin-12 augment the protective immune response in mice orallychallenged with Salmonella dublin. Infect. Immun. 64: 1437–1440.

40. Walters, N., T. Trunkle, M. Sura, and D. W. Pascual. 2005. Enhanced immu-noglobulin A response and protection against Salmonella enterica serovartyphimurium in the absence of the substance P receptor. Infect. Immun. 73: 317–324.

41. Kennedy, P. G., J. Rodgers, B. Bradley, S. P. Hunt, G. Gettinby, S. E. Leeman,C. de Felipe, and M. Murray. 2003. Clinical and neuroinflammatory responses tomeningoencephalitis in substance P receptor knockout mice. Brain 126: 1683–1690.

42. Reinke, E. K., M. J. Johnson, C. Ling, J. Karman, J. Lee, J. V. Weinstock,M. Sandor, and Z. Fabry. 2006. Substance P receptor mediated maintenance ofchronic inflammation in EAE. J. Neuroimmunol. 180: 117–125.

43. Nessler, S., C. Stadelmann, A. Bittner, K. Schlegel, F. Gronen, W. Brueck,B. Hemmer, and N. Sommer. 2006. Suppression of autoimmune encephalomy-elitis by a neurokinin-1 receptor antagonist—a putative role for substance P inCNS inflammation. J. Neuroimmunol. 179: 1–8.

44. Kamon, H., T. Kawabe, H. Kitamura, J. Lee, D. Kamimura, T. Kaisho, S. Akira,A. Iwamatsu, H. Koga, M. Murakami, and T. Hirano. 2006. TRIF-GEFH1-RhoBpathway is involved in MHCII expression on dendritic cells that is critical forCD4 T-cell activation. EMBO J. 25: 4108–4119.

45. Reizis, B., A. Bunin, H. S. Ghosh, K. L. Lewis, and V. Sisirak. 2011. Plasma-cytoid dendritic cells: recent progress and open questions. Annu. Rev. Immunol.29: 163–183.

46. Blasius, A. L., and B. Beutler. 2010. Intracellular toll-like receptors. Immunity32: 305–315.

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