Neuromedin U (NmU), originally isolated from porcine spinal cord and later from other species, is a novel peptide that potently contracts smooth muscle. NmU interacts with two G protein-coupled receptors designated as NmU-1R and NmU-2R. This study demonstrates a potential proinflammatory role for NmU. In a mouse Th2 cell line (D10.G4.1), a single class of high affinity saturable binding sites for 125I-labeled NmU (KD 364 pM and Bmax 1114 fmol/mg protein) was identified, and mRNA encoding NmU-1R, but not NmU-2R, was present. Competition binding analysis revealed equipotent, high affinity binding of NmU isopeptides to membranes prepared from D10.G4.1 cells. Exposure of these cells to NmU isopeptides resulted in an increase in intracellular Ca2+ concentration (EC50 4.8 nM for human NmU). In addition, NmU also significantly increased the synthesis and release of cytokines including IL-4, IL-5, IL-6, IL-10, and IL-13. Studies using pharmacological inhibitors indicated that maximal NmU-evoked cytokine release required functional phospholipase C, calcineurin, MEK, and PI3K pathways. These data suggest a role for NmU in inflammation by stimulating cytokine production by T cells.

Type-2 Th (Th2) cells are CD4+ T cells that secrete cytokines such as ILs 4, 5, and 13 (1). These ILs contribute to a variety of cellular functions including growth, differentiation, and activation of T cells, B cells, and eosinophils and function in immune diseases such as asthma (2). Activation of Th2 cells by Ag recognition through the TCR results in cytokine secretion. Various neuropeptides, including calcitonin gene-related peptide, substance P, and somatostatin, can directly stimulate T cells to produce cytokines (3). An interaction between the CNS and the immune system has been suggested to play a significant role in disease pathology (4).

Neuromedin U (NmU)2 is a neuropeptide widely expressed in the gastrointestinal, genitourinary, and central nervous systems (5). NmU is synthesized from a larger precursor peptide of 174-aa residues and cleaved into 25-aa (human NmU, 25 aa; rat NmU, 23 aa; porcine NmU, 25 aa) and 8-aa (NmU-8; 18–25) biologically active peptides (6, 7).

The biological functions of NmU are diverse. Early characterizations of NmU revealed its potent contractile activity in the rat uterus (6, 7) which were followed by experiments demonstrating contraction of smooth muscle of many organs (8, 9). Several studies indicate that NmU also plays a role in energy homeostasis, (10, 11, 12, 13) regulation of blood pressure (14, 15), and nociception (16, 17). Taken together, these reports indicate a wide variety of functions for NmU and suggest potential mechanisms by which release of signaling molecules from neurons can mediate processes both within the CNS and in peripheral tissues.

Two distinct G protein-coupled receptors (GPCRs) have been identified as NmU-specific receptors, NmU-1R and NmU-2R (also known as FM3 and FM4), from various species (10, 18, 19, 20, 21, 22, 23, 24). Human NmU-1R and NmU-2R share 51% amino acid identity (10). Activation of either NmU-1R or NmU-2R results in Ca2+ release (10, 18, 19, 20, 21, 22, 23, 24, 25) suggesting that these receptors couple to members of the Gq/11 family of G proteins. Although the signaling cascade of these receptors appears to be similar, the expression patterns of NmU-1R and NmU-2R are very different. Human NmU-1R is expressed in gastrointestinal and urogenital systems whereas the expression of NmU-2R is limited to tissues of the CNS. Of particular interest to us is that expression of NmU-1R, but not NmU-2R, has been identified in lung, spleen, and lymphocytes (10, 18, 19), suggesting a potential role in immune responses.

Because RNA encoding NmU-1R has been localized to T cells (18) and the ligand, NmU has been localized to lymphoid cells including dendritic cells, monocytes, and B cells (18), we chose to stimulate a T cell line with NmU and evaluate the resultant changes in transcription and protein synthesis.

Radioligand binding assays identified murine D10.G4.1 cells as Th2-type cells expressing endogenous NmU binding sites. Expression of mRNA by PCR (RT-PCR) indicated the presence of NmU-1R but not NmU-2R. This finding provided us with a physiologically relevant cell line that expressed endogenous NmU-1R which allowed for our evaluation of signaling requiring no transfection for receptor specificity. In addition, the use of a cell line lends itself to microarray analysis because it minimizes background variability. Stimulation of D10.G4.1 cells with NmU resulted in calcium influx as well as increased production of several ILs demonstrated by both microarray and ELISA analysis. Using pharmacological inhibitors we also have shown a requirement of functional phospholipase C (PLC), calcineurin, MEK, and PI3K signaling cascades for maximal NmU-evoked cytokine release. These data are the first to demonstrate a function of NmU and NmU-1R in the immune system.

Human NmU-25, porcine NmU-25, porcine NmU-8, and rat NmU-23 were obtained from Bachem Biochemicals (King of Prussia, PA). Canine NmU-8 was synthesized by California Peptide Research (Napa, CA). Monoiodinated 125I-labeled NmU-25 (human; [125I]NmU-25) was custom synthesized by Amersham (Arlington Heights, IL; specific activity 2000 Ci/mmol). The bicinchoninic acid (BCA) protein assay kit was obtained from Pierce (Rockford, IL). U-73122 and U-73343 were purchased from Biomol (Plymouth Meeting, PA). All Abs were purchased from BD Biosciences/Pharmingen (San Diego, CA). All other reagents were obtained from Sigma-Aldrich (St. Louis, MO).

The Th2 clone D10.G4.1 (D10) cell line was obtained from American Type Culture Collection (ATCC; Manassas, VA) and cultured according to ATCC recommendations. Briefly, cells were grown in RPMI 1640 medium with 2 mM l-glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES, and 1.0 mM sodium pyruvate, and supplemented with: 0.05 mM 2-ME, 10 pg/ml IL-1α, 10% FBS, and 10% rat T-STIM factor with Con A (rat IL-2 culture supplement available from BD Biosciences, cat. no. 354115). Cells were not stimulated with conalbumin or cultured with feeder cells.

Subconfluent monolayers of cells were harvested by scraping the T-150 flasks with Dulbecco’s PBS (DPBS), washed with DPBS, and then pelleted by centrifugation at 500 × g for 10 min. Cell lysis was performed by sonication in buffer containing 5 mM Tris, pH 7.5, 2 mM MgCl2, 1 mM EDTA, 0.2 mg/ml soybean trypsin inhibitor, 4 μg/ml leupeptin, 0.25 mg/ml bacitracin, 1 μM phosphoramidon, and 0.2 mM PMSF and the lysate was centrifuged at 1000 × g for 10 min at 4°C. The supernatants were further centrifuged at 47,000 × g for 20 min at 4°C. Membrane pellets were washed twice by centrifugation in buffer containing 20 mM Tris-HCl, pH 7.4, 5 mM MgCl2, 2 mM Na-EGTA, 0.1 mg/ml bacitracin (buffer B), and resuspended in the same buffer at (5 mg/ml), and stored at −70°C. The protein concentration was measured using the Pierce BCA method with BSA as the standard.

For saturation binding studies, increasing concentrations of 125I-labeled human NmU ([125I]hNmU) were added to membranes (50–100 μg of membrane proteins per milliliter) in a total volume of 200 μl and incubated for 60 min at 25°C. Nonspecific binding was determined in the presence of 1 μM unlabeled hNmU-25. In competition binding studies, membranes (50–100 μg of membrane protein per milliliter) were incubated with increasing concentrations (1 pM-1 μM) of competing ligand and ∼200 pM [125I]hNmU for 60 min at 25°C. The incubations were terminated by addition of 2 ml of cold wash buffer (0.9% NaCl) followed by rapid filtration over Skatron filtermates presoaked in 0.2% polyethyleneimine using a Skatron cell harvester (Skatron Instruments, Tranby, Norway). All binding assays were done in duplicate and each experiment was repeated three to four times. Analyses of all binding data (i.e., the determination of Kd, Bmax, and Ki values) were performed by computer-assisted nonlinear least square fitting using GraphPad Prism (Graphpad Software, San Diego, CA).

Total RNA was treated with DNase I (Ambion, Austin, TX) to remove genomic DNA contamination. The efficiency of the DNase procedure was validated in a standard RT-PCR; assay with a GAPDH primer set using RNA samples that were not subjected to a reverse transcription step. The DNA-free total RNA samples were quantitated using RiboGreen RNA quantitation reagent (Molecular Probes, Eugene, OR). First strand cDNA synthesis was conducted by oligo(dT) priming of 1 μg of each total RNA sample (0.01 M DTT, 0.5 mM each dNTP, 0.5 μg oligo(dT) primer, 40 U of RNaseOUT RNase inhibitor (Invitrogen Life Technologies, Carlsbad, CA), 200 U of Superscript II reverse transcriptase (Invitrogen Life Technologies)). Each sample was plated in duplicate and the equivalent of 25 ng of total RNA was loaded into each well on the 96-well optical microplate.

Quantitative RT-PCR was conducted using an ABI 7700 sequence detector (Applied Biosystems, Foster City, CA) in a 25-ul reaction volume (2.5 mM MgCl2, 0.2 mM dATP, dCTP, dGTP, and dUTP, 0.1 μM each primer, 0.05 M TaqMan probe, 0.01 U of AmpErase uracil-N-glycosylase, 0.0125 U of Amplitaq Gold DNA polymerase (Applied Biosystems, Foster City, CA)). Universal PCR conditions and the following primers designed using Primer Express software (Applied Biosystems) were used.

Genomic standards were included for every gene profiled to generate a standard curve.

Primers are as follows: β-actin 5′-GAGCTATGAGCTGCCTGACG-3′ and 5′-AGTTTCATGGATGCCACAGGA-3′; with probe 5′-6FAM-CATCACTATTGGCAACGAGCGGTTCC-TAMRA-3′; GAPDH 5′-CAAGGTCATCCATGACAACTTTG-3′ and 5′-GGGCCATCCACAGTCTTCTG-3′ with probe

5′-6FAM-ACCACAGTCCATGCCATCACTGCCA-TAMRA-3′; NmU-1R (FM3) 5′-CCTCATGTCTACTCGCTTCCG-3′ and 5′-GATGACAGCACTGGGTTCCA-3′ with probe 5′-6FAM-AGACCTTCCTGCAAGCCCTGGGC-TAMRA-3′; NmU-2R (FM4) 5′-CACGGTTAGCATTGAGCGCT-3′ and 5′-TGTGCTCTCCAGCTTGGCT-3′ with probe 5′-6FAM-CGTGGCCATTGTCCATCCGTTCC-TAMRA-3′.

The cellular functional activity to assess NmU isopeptide effects in D10 cells was Ca2+ mobilization in fura 2-loaded cells. D10 cells (2 × 106 cells/ml) suspended in Krebs-Ringer Henseleit buffer containing 118 mM NaCl, 4.6 mM KCl, 24.9 mM NaHC03, 1 mM KH2PO4, 11.1 mM d-glucose, 5 mM HEPES, pH 7.4, with 1 mM CaCl2, 1.1 mM MgS04, 2.5 mM probenecid, and 0.1% BSA added (buffer A), plus 2 μM fura 2-AM were incubated at 37°C for 30 min. The cells were pelleted by centrifugation at 200 × g for 5 min and resuspended at 2 × 106 cells/ml in buffer A and incubated at 37°C for 15 min. Cells were pelleted and resuspended in buffer A and maintained on ice until used for fluorescence measurements. Intracellular free calcium concentration, [Ca2+]i, was determined in the fura 2-loaded cells by measuring the fluorescence of the entrapped fura 2 using a fluorometer (Johnson Foundation Biomedical Instrumentation Group of the University of Pennsylvania, Philadelphia, PA) as described previously (26).

D10 cells (2 × 106) were plated in 3 ml of growth medium and grown in a 37°C incubator at 5% CO2. The next day (16–18 h later) cells were stimulated with 10 nM NmU (final) or PBS (30 μl of a 100× stock was added to the cells in 3 ml of media). Cells were harvested 0.75, 3, 6, or 12 h poststimulation. Cells were pelleted and RNA was extracted from the pellet using QIAshredder and RNeasy kits according to manufacturer’s instructions (Qiagen, Valencia, CA). Genomic DNA was digested with the DNA Free kit (Ambion, Austin, TX) according to manufacturer’s instructions.

RNA was labeled and hybridized on murine U74Av2 gene chips (Affymetrix, Santa Clara, CA). Total RNA was converted to biotin-labeled cRNA and subsequently hybridized according to manufacturer’s instructions (Affymetrix). Briefly, each sample was processed from 10 μg of total RNA that was first converted to cDNA. All of the cDNA was subsequently converted to biotinylated cRNA by in vitro transcription in the presence of biotin-dUTP. The final cRNA product (20 μg) was fragmented by incubation at 94°C for 30 min in the presence of 40 mM Tris-acetate, pH 8.1, 100 mM potassium acetate, and 30 mM magnesium acetate. Fragmented cRNA (15 μg) was hybridized to the Affymetrix chips. Hybridized chips were washed and then scanned on an Affymetrix GeneChip 3000 confocal scanner. Gene expression data was generated using MAS 5.0 software (Affymetrix). Both total RNA and cRNA samples were checked for quality on a RNA 6000 Nano LabChip using the Bioanalyzer system (Agilent Technologies, Palo Alto, CA).

D10 cells (1 × 106) were preincubated with inhibitors or vehicle (LY294002, U0126, AG-490, U-73122, U-73343 in DMSO; cyclosporin A (CsA) in ethanol; anti-IL-4, anti-IL-4R, or control IgG in PBS) for 1 h at 37°C. Neuromedin U (10 nM final concentration) or vehicle (PBS) was added to the wells and the cells were incubated at 37°C for 6 h. Pelleted cells and the supernatants were stored separately at −70°C before analysis. The concentration of secreted ILs (IL-4, IL-5, IL-6, IL-10, and IL-13) was evaluated by mouse Quantikine ELISA kits according to manufacturer’s instructions (R&D Systems, Minneapolis, MN).

For the microarray study, the average intensity signals for all genes within replicate samples were calculated and log ratios of the averaged signals were used to estimate the fold change in gene expression between treatments. Statistical significance of the difference in expression for a gene between two treatments was assessed by the two-tailed Student t test for unpaired samples.

For the ELISA experiments, GraphPad Prism software (GraphPad Software) was used to determine statistical significance as measured by ANOVA and Dunnett’s posthoc test to compare samples treated with inhibitors to vehicle-treated controls.

Initial characterization of [125I]hNmU (125 pM) binding to D10.G4.1 cell membranes revealed that specific binding at this concentration was >90% of total binding as defined in the presence of unlabeled human NmU (1 μM) (Fig. 1 A). Specific binding was linear with respect to protein concentration up to 50 μg/assay. Time course experiments were conducted with membranes of D10.G4.1 cells at room temperature. Specific binding of [125I]hNmU reached a maximum at 15 min and was maintained up to 3 h (data not shown). All subsequent binding experiments were performed by using a 30-min incubation period. The isotherms of the specific binding of [125I]hNmU were characterized over the concentration range of 50–800 pM. Nonlinear regression analysis of the saturation curves indicated a single class of binding sites (Bmax = 1114 ± 156 fmol/mg protein) displaying high affinity (Kd = 364 ± 5 pM). Nonspecific binding was ∼12% for concentrations near the Kd value.

FIGURE 1.

Mouse D10.G4.1 cells express NmU1R, but not NmU2R. A, Saturation binding of [125I]hNmU in membranes from D10.G4.1 cells. Specific binding is represented. Inset, Scatchard plot of the specific binding. Points shown are from representative experiments performed in duplicate and repeated three times. B, Representative competition curves of NmUs on membrane preparations from D10.G4.1 cells with [125I]hNmU as radioligand. Competition curves with human NmU-25 (▪), rat NmU-23 (□), porcine NmU-25 (▴), porcine NmU-8 (▵), and canine NmU-8 (•). In all cases, maximum binding was expressed as 100%, and nonspecific binding was subtracted. Each point is the mean of duplicate determinations. Similar results were obtained on at least three independent occasions. C, A quantitative PCR experiment in which mRNA from D10.G4.1 cells was extracted and used to evaluate the presence of message encoding NmU receptors. These data demonstrate the presence of NmU1R at 58, 650 copies/50 ng total RNA and failed to detect any message encoding NmU2R.

FIGURE 1.

Mouse D10.G4.1 cells express NmU1R, but not NmU2R. A, Saturation binding of [125I]hNmU in membranes from D10.G4.1 cells. Specific binding is represented. Inset, Scatchard plot of the specific binding. Points shown are from representative experiments performed in duplicate and repeated three times. B, Representative competition curves of NmUs on membrane preparations from D10.G4.1 cells with [125I]hNmU as radioligand. Competition curves with human NmU-25 (▪), rat NmU-23 (□), porcine NmU-25 (▴), porcine NmU-8 (▵), and canine NmU-8 (•). In all cases, maximum binding was expressed as 100%, and nonspecific binding was subtracted. Each point is the mean of duplicate determinations. Similar results were obtained on at least three independent occasions. C, A quantitative PCR experiment in which mRNA from D10.G4.1 cells was extracted and used to evaluate the presence of message encoding NmU receptors. These data demonstrate the presence of NmU1R at 58, 650 copies/50 ng total RNA and failed to detect any message encoding NmU2R.

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Binding of [125I]hNmU binding to D10.G4.1 cell membranes was further characterized in competition studies using human, rat, porcine, and canine NmU isopeptides (Fig. 1,B; Table I). Competition binding revealed that all six isoforms of NmU displaced the radioligand with comparable affinity (Fig. 1,B) (Table I). A similar finding was observed for the competition binding of [125I]hNmU by NmU isopeptides in the membranes from recombinant human NmU receptors. Hill coefficients for NmU peptides were approximated to unity indicating that NmU isopeptides interacted with a single, homogeneous population of binding sites.

Table I.

Binding affinities for NmU isopeptides at D10.G4.1 cellsa

Competing LigandAffinity (Ki), nMnH
Mammalian NmU isopeptides   
 Human NmU-25 0.4 ± 0.1 0.8 
 Rat NmU-23 1.3 ± 0.3 1.2 
 Porcine NmU-25 1.2 ± 0.4 1.2 
 Porcine NmU-8 5.3 ± 1.3 0.9 
 Canine NmU-8 3.5 ± 0.6 0.8 
Competing LigandAffinity (Ki), nMnH
Mammalian NmU isopeptides   
 Human NmU-25 0.4 ± 0.1 0.8 
 Rat NmU-23 1.3 ± 0.3 1.2 
 Porcine NmU-25 1.2 ± 0.4 1.2 
 Porcine NmU-8 5.3 ± 1.3 0.9 
 Canine NmU-8 3.5 ± 0.6 0.8 
a

Ki (affinity) values for each peptide were determined from three to four independent competition binding experiments using membranes isolated from D10.G4.1 cells. Ki values and nH (Hill coefficient) were determined using GraphPad Prism software and represented as the mean ± SEM.

Because NmU is known to activate two distinct GPCRs, we next examined the expression of NmU-1R and NmU-2R mRNAs. Using RT-PCR, we detected message encoding NmU-1R at 58,650 copies per 50 ng of total RNA while NmU-2R message was not measurable (fewer than 10 copies per 50 ng of total RNA). This result suggests that NmU-1R was the receptor mediating the effects of NmU (Fig. 1 C) on D10 cells. No reaction product was seen when the PCR was performed without the initial RT-step, indicating that genomic DNA was not amplified.

Activation of the recombinant NmU-1R receptor has been shown to activate PLC and release intracellular calcium (20, 21). To determine whether stimulation of the endogenous NmU-1R in murine T cells evokes a similar response, we loaded D10.G4.1 cells with fura 2 and measured NmU-mediated changes in intracellular Ca2+ using a fura 2 fluorescence based assay. Basal [Ca2+]i was ∼240 nM in D10.G4.1 cells. Human, rat, and porcine NmU (both C25 and C8 peptides) increased intracellular Ca2+ in a concentration-dependent manner with EC50 values of 4.8, 10.1, 6.0, and 4.8 nM, respectively (n = 2, Fig. 2).

FIGURE 2.

Stimulation of D10.G4.1 cells with NmU isopeptides evoked calcium mobilization response. NmU isopeptide concentration-response curves were run for human NmU-25 (▪), NmU-8 (♦), rat NmU-23 (▾), and porcine NmU-25 (▴). Values presented are the maximal Ca2+concentrations achieved at the indicated concentrations of agonists. Results presented are from a typical experiment; three experiments were conducted.

FIGURE 2.

Stimulation of D10.G4.1 cells with NmU isopeptides evoked calcium mobilization response. NmU isopeptide concentration-response curves were run for human NmU-25 (▪), NmU-8 (♦), rat NmU-23 (▾), and porcine NmU-25 (▴). Values presented are the maximal Ca2+concentrations achieved at the indicated concentrations of agonists. Results presented are from a typical experiment; three experiments were conducted.

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To elucidate the potential pharmacological roles of NmU-1R in murine T cells, we used GeneChips to examine time-dependent changes in mRNA upon stimulation of D10.G4.1 cells with 10 nM NmU (Fig. 3).3 Although this study revealed many significant gene changes as a result of NmU stimulation, we noticed a marked elevation in the levels of Th2 cytokines including IL-4, 5, 6, 10, and 13. There were three distinct temporal expression patterns of NmU-evoked up-regulation of IL mRNAs. IL-4 and IL-13 exhibited modest increases at 45 min with a maximal increase at 3 h poststimulation decreasing toward baseline levels at 6 and 12 h. The mRNA encoding IL-6 and IL-10, however, peaked with maximal increases at 45 min and declined steadily at 3, 6, and 12 h. Finally, IL-5 showed a delayed increase with no change in mRNA level at 45 min but significantly increased expression at 3, 6, and 12 h. (Fig. 3, left panels).

FIGURE 3.

Transcriptome analysis reveals increases in cytokine expression upon treatment of D10.G4.1 cells with NmU. D10.G4.1 cells (2 × 106) cultured under normal growth conditions were stimulated with 10 nM NmU (▪) or vehicle (PBS) (□) for 0, 0.75, 3, 6, or 12 h. The mRNA was extracted and changes in gene expression were measured while protein in the cell supernatant was evaluated by ELISA. Left panels, Relative abundance of mRNA encoding IL-4, IL-5, IL-6, IL-10, and IL-13; right panels, increases in protein as measured by ELISA.

FIGURE 3.

Transcriptome analysis reveals increases in cytokine expression upon treatment of D10.G4.1 cells with NmU. D10.G4.1 cells (2 × 106) cultured under normal growth conditions were stimulated with 10 nM NmU (▪) or vehicle (PBS) (□) for 0, 0.75, 3, 6, or 12 h. The mRNA was extracted and changes in gene expression were measured while protein in the cell supernatant was evaluated by ELISA. Left panels, Relative abundance of mRNA encoding IL-4, IL-5, IL-6, IL-10, and IL-13; right panels, increases in protein as measured by ELISA.

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Because changes in mRNA may not always correlate with changes in protein concentration, we examined secreted IL-4, 5, 6, 10, and 13 concentrations using ELISA. These experiments demonstrated increases in IL-4, 5, 6, 10, and 13 protein that correlated with corresponding increases in mRNA. Concentrations of each IL appeared to increase with time; however, the increase in IL-5 was delayed until the 6-h time point with more significant changes at 12 h poststimulation (Fig. 3, right panels).

Because NmU-evoked increases in cytokine levels were unexpected, we examined signaling pathways by which NmU could promote cytokine secretion. It has been shown previously that stimulation of NmU-1R results in release of inositol phosphates, suggesting activation of PLC (21); therefore, we used the PLC inhibitor U-73122 to test whether NmU-evoked cytokine release was dependent upon PLC. Pretreatment of D10.G4.1 cells with U-73122 resulted in concentration-dependent decreases in IL-4, IL-6, and IL-10 release upon NmU stimulation with significant inhibition of all these cytokines at 1 μM (Fig. 4). However, preincubation of cells with U-73343 resulted in no significant differences from control cells. Thus, functional PLC was required for maximal NmU-stimulated IL secretion.

FIGURE 4.

PLC is required for NmU to evoke increases in cytokine release. D10.G4.1 cells (1 × 106) were pretreated with increasing concentrations of the PLC inhibitor U-73122 or the control compound U-73343 for 1 h before stimulation with NmU (10 nM) (▪) or vehicle (PBS) (□) for 6 h. The cell supernatant was collected and IL concentrations were determined by ELISA. The graphs show the amount of IL released as a percent of maximal release with control compound. ∗, p < 0.05; n = 3–5.

FIGURE 4.

PLC is required for NmU to evoke increases in cytokine release. D10.G4.1 cells (1 × 106) were pretreated with increasing concentrations of the PLC inhibitor U-73122 or the control compound U-73343 for 1 h before stimulation with NmU (10 nM) (▪) or vehicle (PBS) (□) for 6 h. The cell supernatant was collected and IL concentrations were determined by ELISA. The graphs show the amount of IL released as a percent of maximal release with control compound. ∗, p < 0.05; n = 3–5.

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Because IL-4 is known to enhance the development of Th2 cells and cytokine release, we used an Ab to block IL-4 before stimulation with NmU to exclude the possibility that NmU-stimulated IL-4 production and subsequent release could act in an autocrine manner to promote production of other cytokines. Preincubation with an anti-IL-4 Ab had no significant effect on NmU-induced secretion of IL-6 or IL-10, although decreased levels of IL-4 confirmed that the Ab was functional (Fig. 5,A). Similarly, incubation with an Ab to the receptor for IL-4 (IL-4R) also had no effect on NmU-evoked cytokine release (data not shown). It has been demonstrated that stimulation of the IL-4R can activate JAKs. To evaluate a potential role for JAKs in NmU-evoked cytokine release, we incubated cells with AG-490, a JAK inhibitor, before stimulation with NmU. Consistent with the lack of a requirement for IL-4R signaling, NmU-mediated release of the three ILs was not affected by AG-490, suggesting that the JAK pathway was not required (Fig. 5 B). Cumulatively, these results suggest that NmU-evoked increases in cytokine release appear to be independent of IL-4R signaling.

FIGURE 5.

NmU elicits cytokine release independent of the IL-4R signaling pathway. D10.G4.1 cells (1 × 106) were pretreated with Abs to IL-4 or control IgG (A) or increasing concentrations of the JAK inhibitor AG-490 or vehicle (DMSO) (B) for 1 h before stimulation with NmU (10 nM) (▪) or vehicle (PBS) (□) for 6 h. The cell supernatant was collected and IL-4, IL-6, and IL-10 concentrations were determined by ELISA. The graphs show the amount of ILs released normalized to vehicle as a percent of maximal. ∗, p < 0.05, ∗∗, p < 0.01; n = 8–10.

FIGURE 5.

NmU elicits cytokine release independent of the IL-4R signaling pathway. D10.G4.1 cells (1 × 106) were pretreated with Abs to IL-4 or control IgG (A) or increasing concentrations of the JAK inhibitor AG-490 or vehicle (DMSO) (B) for 1 h before stimulation with NmU (10 nM) (▪) or vehicle (PBS) (□) for 6 h. The cell supernatant was collected and IL-4, IL-6, and IL-10 concentrations were determined by ELISA. The graphs show the amount of ILs released normalized to vehicle as a percent of maximal. ∗, p < 0.05, ∗∗, p < 0.01; n = 8–10.

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In this study we demonstrated that stimulation of NmU-1R with NmU resulted in increased intracellular Ca2+. It is well known that Ca2+ can mediate transcription by activation of calcineurin and the dephosphorylation of transcription factors such as the NFAT (27). Preincubation of D10.G4.1 cells with increasing concentrations of CsA, an inhibitor of calcineurin, resulted in a concentration-dependent decrease in NmU-mediated release of these ILs when compared with vehicle (ethanol)-treated control cells (Fig. 6). Significant inhibition of these ILs was achieved at 100 nM CsA.

FIGURE 6.

CsA attenuates NmU-mediated IL release. D10.G4.1 cells (1 × 106) were pretreated with increasing concentrations of the calcineurin inhibitor CsA or vehicle (ethanol) for 1 h before stimulation with NmU (10 nM) (▪) or vehicle (PBS) (□) for 6 h. The cell supernatant was collected and IL-4, IL-6, and IL-10 concentrations were determined by ELISA. The graphs show the amount of ILs released normalized to vehicle as a percent of maximal. ∗, p < 0.05, ∗∗, p < 0.01; n = 8–10.

FIGURE 6.

CsA attenuates NmU-mediated IL release. D10.G4.1 cells (1 × 106) were pretreated with increasing concentrations of the calcineurin inhibitor CsA or vehicle (ethanol) for 1 h before stimulation with NmU (10 nM) (▪) or vehicle (PBS) (□) for 6 h. The cell supernatant was collected and IL-4, IL-6, and IL-10 concentrations were determined by ELISA. The graphs show the amount of ILs released normalized to vehicle as a percent of maximal. ∗, p < 0.05, ∗∗, p < 0.01; n = 8–10.

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The activation of PI3K has also been reported to be an important signaling mechanism in T cells (28). To examine the requirement of PI3K for NmU-induced cytokine release, D10.G4.1 cells were preincubated with LY294002, an inhibitor of PI3K. Compared with vehicle (DMSO)-treated cells, samples preincubated with LY294002 exhibited a concentration-dependent inhibition of NmU-evoked IL release, suggesting that PI3K was required for NmU to elicit cytokine secretion from D10.G4.1 cells (Fig. 7).

FIGURE 7.

PI3K is required for NmU-stimulated increases in cytokine release. D10.G4.1 cells (1 × 106) were pretreated with increasing concentrations of the PI3K inhibitor LY294002 or vehicle (DMSO) for 1 h before stimulation with NmU (10 nM) (▪) or vehicle (PBS) (□) for 6 h. The cell supernatant was collected and IL-4, IL-6, and IL-10 concentrations were determined by ELISA. The graphs show the amount of ILs released normalized to vehicle as a percent of maximal. ∗, p < 0.05, ∗∗, p < 0.01; n = 5–10.

FIGURE 7.

PI3K is required for NmU-stimulated increases in cytokine release. D10.G4.1 cells (1 × 106) were pretreated with increasing concentrations of the PI3K inhibitor LY294002 or vehicle (DMSO) for 1 h before stimulation with NmU (10 nM) (▪) or vehicle (PBS) (□) for 6 h. The cell supernatant was collected and IL-4, IL-6, and IL-10 concentrations were determined by ELISA. The graphs show the amount of ILs released normalized to vehicle as a percent of maximal. ∗, p < 0.05, ∗∗, p < 0.01; n = 5–10.

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MAPK pathways are often stimulated by GPCR activation. To evaluate the requirement of MAPK for NmU-evoked cytokine release, we preincubated D10.G4.1 cells with U0126, a MEK 1 and 2 inhibitor (29). The addition of U0126 resulted in a concentration-dependent decrease in the ability of NmU to elicit IL secretion compared with control (DMSO)-treated cells (Fig. 8) bringing cytokine release back to control levels at 50 μM U0126. These data suggest that the MAPK signaling cascade is essential for NmU-mediated increases in cytokine release.

FIGURE 8.

The MEK inhibitor U0126 blocks NmU-evoked cytokine release. D10.G4.1 cells (1 × 106) were pretreated with increasing concentrations of the MEK inhibitor U0126 or vehicle (DMSO) for 1 h before stimulation with NmU (10 nM) (▪) or vehicle (PBS) (□) for 6 h. The cell supernatant was collected and IL-4, IL-6, and IL-10 concentrations were determined by ELISA. The graphs show the amount of ILs released normalized to vehicle as a percent of maximal. ∗, p < 0.05, ∗∗, p < 0.01; n = 3–8.

FIGURE 8.

The MEK inhibitor U0126 blocks NmU-evoked cytokine release. D10.G4.1 cells (1 × 106) were pretreated with increasing concentrations of the MEK inhibitor U0126 or vehicle (DMSO) for 1 h before stimulation with NmU (10 nM) (▪) or vehicle (PBS) (□) for 6 h. The cell supernatant was collected and IL-4, IL-6, and IL-10 concentrations were determined by ELISA. The graphs show the amount of ILs released normalized to vehicle as a percent of maximal. ∗, p < 0.05, ∗∗, p < 0.01; n = 3–8.

Close modal

The present study reports the identification of endogenous, functional NmU receptors in D10 cells. These cells are from the Th cell clone of type 2 phenotype D10.G4.1. As such, the characterization of these cell lines may facilitate a greater understanding of the biochemical and pharmacological characteristics of NmU and its cognate receptor(s), elucidating the physiological role(s) of this novel peptide. Our results suggest that D10 cells have single site high affinity NmU binding with high expression of NmU receptors. Scatchard plots as well as the Hill coefficients for receptor binding and functional activity were close to unity, suggesting the presence of a single population of NmU receptors. Using RT-PCR, we demonstrated expression of mRNA transcripts for NmU-1R in D10.G4.1 cells. The binding affinity of NmU isopeptides in D10.G4.1 cell membranes was in close agreement with the affinities observed for recombinant NmU receptors. Exposure of these cells to NmU isopeptides resulted in an increase in intracellular Ca2+ release (EC50 6 nM), which depended on PLC activation. In addition, stimulation of D10.G4.1 cells with NmU resulted in increased synthesis and secretion of IL-4, 5, 6, 10, and 13, demonstrating for the first time that NmU can affect immune cell function.

Various inhibitors were used to evaluate signaling pathways by which NmU stimulates cytokine release. It has been shown previously that NmU activates PLC which catalyzes the conversion of phosphoinositide 4,5-bisphosphate to diacylglycerol (DAG) and inositol 1,4,5 Tris-phosphate (IP3). Here, we demonstrated that inhibition of PLC by U-73122 inhibited NmU-evoked cytokine release. If the signaling cascades stimulated by NmU follow a classic paradigm of GPCR signaling, inhibition of the G protein effector PLCβ, a relatively early event in the sequence initiated by agonist binding the receptor, should prevent synthesis of mRNA and cytokine release. Therefore, it is not surprising that a PLC inhibitor blocked these responses. These data support that the NmU-NmU-1R pathway stimulates a classic Gq/11 signaling cascade.

PLC catalyzes the generation of DAG and IP3, and IP3 subsequently elicits Ca2+ release from intracellular stores (30). A well-characterized pathway of T cell activation by the TCR also results in Ca2+ release from intracellular stores, calmodulin binding, and stimulation of the protein phosphatase calcineurin (31). Dephosphorylation of members of the NFAT family by calcineurin allows NFATs to enter the nucleus and stimulate transcription (32). CsA, an immunosuppressive agent, can block the calcineurin signaling cascade (33). Here, we showed that stimulation of D10.G4.1 cells with NmU results in an increase in intracellular Ca2+ and that preincubation with CsA attenuates the ability of NmU to evoke IL release. Thus, it appears that NmU- and TCR-mediated T cell activation share the requirement of functional calcineurin in the synthesis and release of cytokines.

An additional signaling cascade in T cells is the activation of JAKs by cytokine receptors. The binding of IL-4 to its receptor can result in the activation of JAKs, which phosphorylate STATs (34). Preincubation with AG-490, a JAK2 and 3 inhibitor, blocks IL-4-evoked IL-4 synthesis in T cells (35). Here, we demonstrated that preincubation of AG-490 does not attenuate NmU-mediated IL release. In addition, NmU-evoked cytokine secretion is not blocked by Abs to either IL-4 or the IL-4R, providing further evidence that NmU-mediated release of IL-4, IL-6 and IL-10 occurs independently of the IL-4 signaling cascade.

MAPKs mediate various signaling cascades in T cells. In particular, the ERK pathway has been reported to promote Th2 cell differentiation (36). TCR activation stimulates the Ras-MAPK (likely MEK) pathway, (37) which enhances IL-4R signaling (38). An ERK-dependent component was previously revealed in TCR-stimulated production of IL-4, 5, and 10 (39). We evaluated the requirement for the MEK-ERK pathway in NmU-evoked cytokine release by inhibition of MEK1, 2 by U0126. Preincubation of D10.G4.1 cells with U0126 prevented NmU-mediated cytokine release, consistent with a requirement for MAPK activation. In addition, because inhibition of JAK2 and 3 with AG-490 and of IL-4R by Abs failed to alter NmU-evoked cytokine release, it is unlikely that MEK-enhanced IL-4R signaling was involved in the NmU response.

G protein β-γ subunits stimulated by GPCR agonists activate PI3Kγ, promoting cytokine transcription (28). T cells from PI3Kγ-deficient mice exhibit diminished cytokine production (40). NmU-mediated cytokine release requires PI3K as demonstrated by the sensitivity of the response to the PI3K inhibitor LY294002. However, it is not clear whether PI3K is activated by β-γ subunits that interact with NmU-1R or whether it is activated by an alternate signaling cascades such as Ras.

Two alternate conclusions can be drawn from the pathway analysis of NmU-evoked cytokine release. The first is that binding of NmU to NmU-1R initiates a linear series of events starting with G protein activation and ending with transcription and production of ILs. Inhibition of any enzyme in this linear cascade ought to prevent the propagation of the signal and therefore diminish NmU-evoked IL synthesis. A second hypothesis is that NmU binding to the NmU-1R stimulates several signaling cascades acting in parallel. The convergence of these signals allows transcription of ILs, but the inhibition of any member of the signaling network would prevent IL synthesis. In support of this hypothesis is that NFAT (activated via the Ca2+/calcineurin pathway) and AP1 (activated via the MAPK pathway) cooperate and bind target DNA sequences to initiate transcription of ILs (41). Another point where stimulation of distinct signaling cascades could occur would be at the small G protein Ras. As mentioned previously, Ras stimulates PI3K and activates the Raf-MEK-ERK signaling pathway. Ras could also be activated by one of several guanine exchange factors (GEFs) including RasGRP, which is stimulated by DAG released upon GPCR-evoked PLC activation (42).

The expression of NmU is widespread with significant levels found in the pituitary, brain and spinal cord, genitourinary tract, and the gastrointestinal tract (5). The vast expression pattern of NmU suggests that this peptide may promote inflammatory responses in many tissues. Another example of a proinflammatory neuropeptide is substance P, which stimulates cytokine release from various leukocyte populations (43). In addition to those tissues noted above, NmU has been localized to immune cells including monocytes, B cells, and dendritic cells, all of which function as APCs, initiating T cell activation (18). Thus, the localization of NmU to APCs suggests that the signaling cascade evoked by NmU through NmU-1R represents a new mechanism by which the immune system itself enhances inflammatory responses.

We have demonstrated a pathway by which NmU can enhance cytokine secretion and therefore amplifies the immune response. It remains to be determined in which physiological and pathological situations NmU signaling is involved. The availability of specific antagonists to NmU-1R and NmU-2R as well as knockout mice would be extremely useful tools in these endeavors and may reveal novel targets for pharmacological inhibition of Th2 signaling, an important component of inflammatory diseases such as asthma.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

2

Abbreviations used in this paper: NmU, Neuromedin U; GPCR, G protein-coupled receptor; PLC, phospholipase C; hNmU, human NmU; [125I]NmU, 125I-labeled NmU; [125I]hNmU, 125I-labeled hNmU; CsA, cyclosporin A; DAG, diacylglycerol; IP3, inositol 1,4,5-tris phosphate.

3

The results of the microarray experiment have been submitted to the Gene Expression Omnibus (GEO) data repository under the accession number GSE1791. http://www.ncbi.nlm.nih.gov/geo/

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