QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.108.138719v1 327/1/215    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Amantini, C. Articles by Santoni, G. Search for Related Content PubMed PubMed Citation Articles by Amantini, C. Articles by Santoni, G.

INFLAMMATION, IMMUNOPHARMACOLOGY, AND ASTHMA

Thiorphan-Induced Survival and Proliferation of Rat Thymocytes by Activation of Akt/Survivin Pathway and Inhibition of Caspase-3 Activity

Department of Experimental Medicine and Public Health, University of Camerino, Camerino, Italy

Received for publication March 7, 2008
Accepted July 9, 2008.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
The activity of substance P (SP) in the rat thymus seems to be tightly controlled by its bioavailability. In this study, we provide evidence for the expression of the SP-degrading enzyme, neutral endopeptidase (NEP)/CD10, by rat thymocyte subsets, and we illustrate its involvement in the in vivo SP/neurokinin-1 receptor (NK₁R)-mediated regulation of thymocyte survival and proliferation. NEP/CD10 was expressed at both mRNA and protein levels on a substantial portion (45.5%) of CD5⁺ thymocytes, namely on the CD4⁺CD8⁺ (double positive; DP) and CD4⁺ subsets. Continuous administration of thiorphan, a specific NEP/CD10 inhibitor, by means of miniosmotic pumps, enhanced rat thymocyte preprotachykinin-A (PPT-A) and NK₁R mRNA expression as well as SP and NK₁R protein levels in an NK₁R-dependent manner. Thiorphan increased CD10⁺CD4⁺ and CD10⁺DP thymocyte numbers, and an NK₁R antagonist, (S)1-{2-[3(3-4-dichlorophenyl)-1-(3-iso-propoxyphenylacetyl)-piperidine-3-yl]ethyl}-4-pheny-1-azoniabicyclo[2.2.2]octane, chloride (SR140333), abrogated these stimulatory effects. In addition, the NEP/CD10 inhibitor stimulated interleukin (IL)-2 production, IL-2 receptor chain expression, and concanavalin A-induced proliferation of CD5⁺ thymocytes, and it inhibited spontaneous and NK₁R-dependent thymocyte apoptosis. The thiorphan-protective antiapoptotic and proliferative effects involved the activation of Akt serine-threonine kinase, subsequent up-regulation of survivin mRNA, down-regulation of procaspase-3 mRNA levels, and suppression of caspase-3 activity, which were inhibited by SR140333 and mimicked by exogenous SP administration. Overall, our findings suggest that by controlling SP availability, NEP/CD10 negatively regulates thymocyte homeostasis and development.

The neutral endopeptidase (NEP)-24.11 (EC 3.4.24.11 [EC] ), also known as enkephalinase, neprilysin, common acute lymphoblastic leukemia antigen, or CD10 (Turner and Tanzawa, 1997), is the prototype of a family of membrane-bound zinc-dependent endopeptidases that regulate the physiological action of a variety of peptides by lowering their extracellular concentration available for receptor binding. Molecular cloning of NEP/CD10 revealed that it is a type II integral membrane protein consisting of a short NH₂-terminal cytoplasmic domain and a large extracellular domain that contains the active site of the enzyme. It was first isolated as a major 90 to 100-kDa glycoprotein from the renal brush border membrane of rabbits (Kerr and Kenny, 1974).

NEP/CD10 is expressed on early normal T- and B-cell progenitors (Shipp and Look, 1993) and on the majority of acute lymphoblastic leukemias and lymphoid malignancies with immature phenotypes (Ritz et al., 1981). Unfractionated human thymocytes express significant levels of NEP/CD10 (Mari et al., 1994; Guérin et al., 1997); however, no data concerning the expression of NEP/CD10 in distinct thymocyte subpopulations have been provided so far.

Physiological substrates for NEP/CD10 are represented by small peptides such as enkephalins, f-Met-Leu-Phe, angiotensins, bradykinin, endothelin-1 (ET-1), thymopoietin (TMPO), bombesin-like peptides, β calcitonin gene-related peptide (CGRP), and substance P (SP) (Shipp and Look 1993). Among these peptides, SP is one of the most kinetically favorable substrates (Matsas et al., 1984). Thus, NEP/CD10 degrades SP by hydrolysis of the Gln⁶-Phe⁷, Phe⁷-Phe⁸, and Gly⁹-Leu¹⁰ bonds, generating different SP fragments.

SP is involved in the modulation of a variety of immune responses in animal models of neurogenic inflammation (Grady et al., 2000) and immune-based disease (Keeble et al., 2005; Michalski et al., 2007). The biological activities of SP are mediated by seven transmembrane-spanning G protein-coupled receptors named neurokinin receptors (NK₁R, NK₂R, and NK₃R), with NK₁R showing the higher affinity for SP (Maggi, 1995).

SP in the rat thymus not only derives from sensory nerve fiber-mediated axonal transport and release in the thymic microenvironment (Jurjus et al., 1998), but it is also synthesized and spontaneously released by thymocytes (Santoni et al., 2002). In this regard, we have provided evidence for the expression of the SP precursor preprotachykinin-A (PPT-A) mRNA, mature SP, and its NK₁R by CD4⁺ and CD4⁺CD8⁺ (double positive; DP) rat thymocytes.

The involvement of NEP in terminating the proinflammatory and immunomodulatory effects of SP has been studied by using selective inhibitors and NEP knockout mice. Highly specific NEP inhibitors, such as thiorphan and phosphoramidon, have been used as pharmacological tools, and they were found to potentiate the activity of SP (Roques et al., 1980; Matsas et al., 1984; Okamoto et al., 1994). NEP^-/- mice are highly sensitive to bacterial endotoxins, endotoxic shock, and intestinal inflammation (Lu et al., 1995), and they show augmented allergic contact dermatitis, which is abrogated by treatment with the NK₁R antagonist SR140333 (Scholzen et al., 2001). In addition, these mice showed subtle differences in lymphoid development (Lu et al., 1995). SP can also modulate thymocyte survival pathways (Santoni et al., 2002), although the signaling pathways by which SP affects cell survival are still poorly elucidated.

Akt/protein kinase B is a serine-threonine kinase that mediates cell survival in various cell types including thymocytes (Coffer et al., 1998); growth factors, cytokines, and hormones cause phosphatidylinositol-3 kinase (PI3-K) activation and generate the membrane phospholipid phosphatidylinositol-3,4,5-trisphosphate that recruits Akt to the membrane, where it becomes phosphorylated at Thr308 and Ser473 residues. Once activated, Akt phosphorylates survival-mediated targets, including Bcl-2 family members and caspases, thus inhibiting apoptosis and promoting cell survival (Yang et al., 2004).

Survivin is the smallest member of the recently described inhibitor of apoptosis (IAP) gene family involved in the regulation of mitosis and cell death (Ambrosini et al., 1998). Like other IAP family members, survivin inhibits processing of procaspase-3 and procaspase-7, and it specifically binds the active form of both caspases through a baculovirus IAP repeat domain. The aim of this study was to evaluate the expression of NEP/CD10 on distinct subsets of rat thymocytes at the mRNA and protein levels and to assess its involvement on thymocyte survival and proliferation by the analysis of the Akt/survivin pathway and caspase-3 activity.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Animals. Specific pathogen-free male Wistar rats (Charles River Italica, Calco, Italy) were used for all experiments. Newborn rats were housed at a constant temperature and humidity with a 12-h light/dark cycle, and they were given food pellets and tap water ad libitum. Age-matched rats were acclimatized before distribution into the experimental groups. No changes on heart rate were found in thiorphan-treated rats compared with untreated and vehicle-treated rats (data not shown). All procedures were done in accordance with the European Communities Council Directive and the guidelines set forth in the National Institutes of Health Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, 1996).

Thiorphan, SP, and NK₁R Antagonist (SR140333) Administration. Thiorphan N-[(RS)-2-benzyl-3-mercaptopropanoyl]-glycine (Sigma-Aldrich, St. Louis, MO), a specific neutral peptidase inhibitor (Roques et al., 1980) diluted in 10% ethanol, was continuously administered for 7 days alone (2 mg/kg) or in combination with the NK₁R antagonist SR140333 (kindly provided by sanofi-aventis, Bridgewater, CT) (Edmonds-Alt et al., 1993) by means of miniosmotic pumps (Alza, Palo Alto, CA) subcutaneously implanted in normal rats at day 21 after birth as described previously (Santoni et al., 1999). The starting thiorphan concentration in the pump chamber was 7.3 x 10^-3 M, with a mean pumping rate of 1.06 ± 0.03 µl/h.

SR140333, diluted in distilled water, was administered alone (20 mg/ml) or in combination with thiorphan as described previously (Santoni et al., 1999). SP (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met) (1 mg/ml), 98.6% pure as assessed by high-performance liquid chromatography (Peninsula Laboratories, Belmont, CA), was diluted in distilled water and administered as described previously (Santoni et al., 1999). As the control group, normal rats received implants filled with the respective vehicles. Five animals from each group were sacrificed at day 28 after birth. Because no major differences were found between the untreated versus thiorphan, SP, or SR140333 vehicle in all of the experiments, for sake the simplicity we use the term vehicle to refer to all of the vehicles used.

We chose to deliver SP, SR140333, and thiorphan by a continuous route of administration, because this modality closely mimics the pathophysiological state observed in immune-mediated diseases, wherein sensory nerves and cells are presumably continuously stimulated to release tachykinins, such as SP (Maa et al., 2000).

Whole blood was collected for measurement of the plasma basal levels of corticosterone. Plasma corticosterone levels were determined by radioimmunoassay (MP Biomedicals, Irvine, CA). Corticosterone concentrations are expressed as nanogram per milliliter of plasma. The sensitivity was 12.5 ng/ml plasma. Thiorphan, administered as described above, did not affect basal plasma levels of corticosterone, compared with the control groups (thiorphan-treated rats, 20.1 ± 1.5 ng/ml; untreated rats, 19.3 ± 2.0 ng/ml; vehicle-treated rats, 20.5 ± 1.7 ng/ml).

Thymus Cell Preparation. Thymi from untreated rats or rats given thiorphan, SR140333, thiorphan plus SR140333, SP, or their respective vehicles were teased, and cellular debris were removed by intense washing. Thymocytes were isolated by centrifugation on Ficoll-Hypaque (Lymphoprep; Nycomed, Oslo, Norway) gradients, washed twice in RPMI 1640 medium (Flow Laboratories, Irvine, Scotland, UK), and counted and diluted at appropriate concentrations in RPMI 1640 medium containing 10% heat-inactivated fetal calf serum (Euroclone, Devon, UK), 2 mM L-glutamine, 100 IU/ml penicillin, and 100 µg of streptomycin. Cell purity, as assessed by immunofluorescence and fluorescent-activated cell sorting (FACS) analysis using mouse anti-rat CD5 (monoclonal antibodies; mAbs), was routinely 99%.

Antibodies. We used the following mouse mAbs: phycoerythrin (PE)-conjugated and purified anti-rat CD5 (clone OX19), fluorescein-isothiocyanate (FITC)-conjugated anti-rat CD25 (clone OX39), PE-conjugated anti-rat CD8 (clone OX8), and FITC-conjugated anti-rat CD4 (clone W3/25) (obtained from BD Pharmingen, San Diego, CA) and anti-rat -tubulin (Millipore Bioscience Research Reagents, Temecula, CA). The following affinity-purified polyclonal antibodies were used: goat anti-rat SP raised against the mature peptide, goat anti-rat NK₁R, rabbit anti-rat NEP/CD10, rabbit anti-rat Akt, rabbit anti-rat phospho-Akt (Ser473) (Cell Signaling Technology Inc., Danvers, MA), and rabbit anti-rat caspase-3 (Calbiochem-Novabiochem, San Diego, CA). The FITC-conjugated rabbit anti-goat (RAG), FITC-conjugated goat anti-rabbit (GAR), and horseradish peroxidase (HRP)-conjugated RAG were purchased from Biomeda (Foster City, CA). The HRP-conjugated donkey anti-rabbit and RAG-biotinylated and tricolor-conjugated streptavidin were obtained from GE Healthcare (Little Chalfont, Buckinghamshire, UK), and the HRP-conjugated sheep anti-mouse was obtained from Invitrogen (Carlsbad, CA). PE-mouse IgG1, FITC-mouse IgG1 (BD Biosciences), as well as goat or rabbit serum (Cappel Laboratories, Durham, NC), were used as negative controls.

Immunofluorescence and Flow Cytometric Analysis. To determine the expression of NEP/CD10 on rat thymocytes (99% CD5⁺ pure), 1 x 10⁶ cells from untreated, vehicle-, thiorphan-, SR140333-, or thiorphan plus SR140333-administered rats were stained with the rabbit anti-rat NEP/CD10 polyclonal antibody (Ab). Normal rabbit serum was used as a negative control. After 30 min at 4°C, the cells were washed twice and labeled with FITC-conjugated GAR (1:20 dilution). The expression of NEP/CD10 on distinct rat thymocyte subpopulations was evaluated by tricolor immunofluorescence and flow-cytometric analysis using PE-conjugated anti-rat CD8, FITC-conjugated anti-rat CD4, and rabbit anti-rat CD10. In brief, 1 x 10⁶ thymocytes from untreated, vehicle-, thiorphan, SR140333, or thiorphan plus SR140333-administered rats were labeled with PE-conjugated anti-rat CD8, FITC-conjugated anti-rat CD4, and rabbit anti-CD10 followed by biotin-conjugated GAR IgG (1:50 dilution) and tricolor-conjugated streptavidin. PE mouse IgG1 and FITC mouse IgG2a were used as negative controls.

To analyze the expression of NK₁R and membrane-bound SP on thymocytes, highly purified CD5⁺ cells from untreated, vehicle-, thiorphan, SR140333, or thiorphan plus SR140333-administered rats were evaluated by staining with anti-SP or anti-NK₁R polyclonal Ab (1:50 dilution) followed by FITC-conjugated RAG (1:20 dilution).

Finally, CD5⁺ thymocytes from untreated, vehicle-, thiorphan-, SR140333-, thiorphan plus SR140333-administered rats were double labeled with FITC-conjugated anti-CD5 and PE-conjugated anti-CD25 for 30 min at 4°C and then washed twice. Thymocytes were then analyzed for relative fluorescence intensity. For tricolor cytofluorometric analysis, electronic compensation was used between green and orange and between orange and red fluorescences to remove spectral overlap. The percentage of positive cells was determined over 10,000 events, and samples were analyzed on a FACScan cytofluorimeter (BD Biosciences, San Jose, CA) by using CellQuest software (BD Biosciences). Fluorescent intensity was expressed in arbitrary units on a logarithmic scale.

Western Blot. Lysates that were obtained from untreated rat thymocytes were resuspended in 0.2 ml of radioimmunoprecipitation assay (0.1% Nonidet P-40, 1 mM CaCl₂, 1 mM MgCl₂, 0.1% sodium azide, 1 mM phenylmethylsulfonyl fluoride, 0.03 mg/ml aprotinin, and 1 mM NaVO₄). Samples were separated on 7% SDS-polyacrylamide gel, transferred to Immobilon-P membranes (Millipore Corporation, Billerica, MA), and blotted with rabbit anti-CD10/NEP (1:400 dilution), and then incubated with the HPR-conjugated anti-rabbit (1:10000) Ab. For Akt detection, thymocytes from untreated, vehicle-, thiorphan-, SR140333-, thiorphan plus SR140333-, or SP-administered rats were resuspended for 15 min in serum-free RPMI 1640 medium, lysed, resolved by 8% SDS-polyacrylamide gel electrophoresis (PAGE), and transferred. Membranes were immunoblotted with anti-Akt Ab (1:500) and anti-phospho-Ser473-Akt Ab (1:1000) followed by incubation with the HRP-conjugated anti-rabbit (1:10000) Ab. For caspase-3 activation, proteins were separated on 14% SDS-polyacrylamide gels, transferred overnight at 20 V, and incubated for 2 h at room temperature with a rabbit anti-caspase-3 Ab (1:500 dilution) followed by donkey HRP-conjugated anti-rabbit (1:10000 dilution) Ab.

Immunoreactivity and densitometric analysis were detected with enhanced chemiluminescence (ECL; Amersham Life Sciences) and analyzed by ChemiDoc and QuantityOne software (Bio-Rad, Hercules, CA). All experiments were performed at least twice using different cell lysates with similar results. Each sample was compared with its control (-tubulin) for the purpose of quantification.

Proliferation Assay. A total of 2 x 10⁵ thymocytes from untreated, vehicle-, thiorphan-, SR140333-, or thiorphan plus SR140333-administered rats were dispersed into tissue cultured-treated plastic, 96-well, round-bottomed microtiter plates (Corning Life Sciences, Lowell, MA) and cultured for 72 h with 5.0 µg/ml concanavalin A (ConA; Sigma-Aldrich) in a total volume of 200 µl of culture medium at 37°C in a 5% humidified atmosphere. Cell proliferation was evaluated by pulsing the cells with 0.5 µCi/well tritiated thymidine ([³H]TdR) (6.7 Ci/mmol; PerkinElmer Life and Analytical Sciences, Waltham, MA) during the last 6 h of the 72-h culture. Incorporation of [³H]TdR was measured by a standard liquid scintillation counting technique after harvesting the cells with the Dynatech harvester (International PBI, Milan, Italy). All cultures were run at least in quadruplicate. Data are expressed as stimulation index and calculated as follows: [³H]TdR uptake in ConA-stimulated cells/unstimulated cells.

Interleukin-2 Assay. Interleukin (IL)-2 levels were measured in the supernatants of unstimulated or 24-h ConA- (5 µg/ml) stimulated thymocyte cultures from untreated, vehicle-, thiorphan-, SR140333-, or thiorphan plus SR140333-administered rats by a quantitative sandwich enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Minneapolis, MN). Results are expressed in picograms per milliliter as the mean concentration ± S.D. of culture supernatants measured in duplicate, and data were pooled from three separate experiments.

Apoptosis Assays. Apoptosis of thymocytes from untreated, vehicle-, thiorphan-, SR140333-, or thiorphan plus SR140333-administered rats was evaluated by biparametric cytofluorimetric analysis. In brief, 10⁶ thymocytes were resuspended in 0.2 ml of binding buffer (10 mM HEPES/NaOH, pH 7.4, 150 mM NaCl, 5 mM KCl, 1 mM MgCl₂, and 1.8 mM CaCl₂) in the presence of 5 µl of FITC-Annexin V (AnnV) (Bender MedSystem, Vienna, Austria) and then incubated for 10 min at room temperature in the dark. Cells were washed, resuspended in 0.2 ml of binding buffer containing 10 µl of propidium iodine (PI) (20 µg/ml in phosphate-buffered saline) (Invitrogen) to discriminate apoptotic from necrotic cells, and then analyzed on a FACScan cytofluorimeter using the CellQuest software.

RNA Isolation and Reverse Transcription. mRNA was extracted by Ficoll-purified thymocytes from untreated, vehicle-, SR140333-, thiorpan plus SR140333-, and thiorphan-administered rats and from rat brain tissues (frontal cortex) used as a positive control (Pollard et al., 1989), using the Quick Messenger RNA Direct Kit (Talent, Trieste, Italy). The mRNA samples were resuspended in diethyl pyrocarbonate water (Sigma-Aldrich, Milano, Italy), and their concentration and purity were evaluated by A₂₆₀ measurement. mRNA samples (200 µg) were subjected to reverse transcription using the High-Capacity cDNA Archive Kit [room temperature buffer, deoxynucleotide triphosphate mixture, random primers, MultiScribe reverse transcriptase, and RNase inhibitor (Applied Biosystems, Monza, Italy)]. Two microliters of resulting cDNA products was used as template for polymerase chain reaction (PCR) analysis.

Qualitative and Quantitative Real-Time PCR Analysis. Qualitative PCR was performed using a GeneAmp 5700 Sequence Detection System (Applied Biosystems). The following forward and reverse primers specific for rat NEP were used: 5'-CCCCGCCGGCATTT-3' and 5'-GCCCCCATAGTTCAATGAGTTG-3'. PCR products were analyzed by electrophoresis in 2% ethidium bromide-stained agarose gel visualized by UV transilluminator and acquired by using ChemiDoc.

Real-time (RT) PCR was performed using an iQ5 Multicolor Real-Time PCR Detection System (Bio-Rad). The reaction mixture contained the TaqMan Universal PCR Master Mix (Applied Biosystems) and TaqMan primers and probe sets. TaqMan forward and reverse primers and probe sets were designed from sequences in the GenBank database by Primer Express (Applied Biosystems): PPT-A, 5'-CGCACTATCTATTCATCTCCATCTG-3', 5'-CATTCCTCATAGCGCACATTTTT-3', and 5'-TCCGCGAGCAGTGAGCGGTAAAA-3'; NK₁R, 5'-GCAGGTGAGAATGCTGTTCCT-3', 5'-TTCACGGATGTCACATGAAGCT-3', and 5'-CGTCTCAGATGTGACTGGAAGCGA-3'; βCGRP, 5'-TATGATGGTGTCTCCTGCTG-3', 5'-GATATCACGTCTCCCTGAGG-3', and 5'-TCACCAGAGCTGCTACCACCGTC-3'; ET-1, 5'-CAGAGACCAGAAGTTGATACAC-3', 5'-CGGACAGATGTTCTTGCTAAG-3', and 5'-ACCGAGCACATTGACTACAGAGCC-3'; TMPO, 5'-ACCAGAACATGGACCAGAAC-3', 5'-GAGGAAAGCACTTCAGATCTAAG-3', and 5'-CCCACACGCCTCACGGACCA-3'; procaspase-3, 5'-AATTCAAGGGACGGGTCATG-3', 5'-GCTTGTGCGCGTACAGTTTC-3', and 5'-TTCATCCAGTCACTTTGCGCCATGC-3'; survivin, 5'-CCACTTGTCCCAGCTTTCCA-3', 5'-GAAATGTCACGGTCTCCTGTAAGA-3', and 5'-CCTGCCTGGCCGCCTTGGT-3'; β-actin, 5'-CGTGAAAAGATGACCCAGATCA-3', 5'-CACAGCCTGGATGGCTACGT-3', and 5'-TTGAGACCTTCAACACCCCAGCCATG-3'; and glyceraldehyde-3-phosphate dehydrogenase, 5'-CGGATTTGGCCGTATTGG-3', 5'-CAATGTCCACTTTGTCACAAGAGAA-3', and 5'-CGCCTGGTCACCAGGGCTG-3'.

The PPT-A primer pair identifies all four isoforms of the PPT-A gene transcript. Each PCR amplification consists of heat activation for 2 min at 50°C and for 10 min at 95°C followed by 45 cycles of 94°C for 20 s, 55°C for 20 s, and 72°C for 30 s. All controls and samples were assayed in duplicate in the same plate. The measurement of glyceraldehyde-3-phosphate dehydrogenase and β-actin mRNA levels of the same samples, performed in the same plate, was used as a control to normalized the mRNA contents. PPT-A, NK₁R, CGRP, ET-1, TMPO, procaspase-3, and survivin mRNA levels were expressed as relative -fold of that of the corresponding control as indicated in the figure legends. Statistical analysis was carried out on all of the TaqMan PCR data.

View larger version (23K): [in this window] [in a new window]   Fig. 1. Expression of CD10/NEP mRNA and protein on distinct rat thymocyte subsets. A, NEP/CD10 mRNA levels on rat thymocytes (>99% CD5+ cells) and brain tissue were evaluated by PCR assay. Data shown are representative of three separate experiments. B, lysates from thymocytes and brain tissue were separated on 7% SDS-PAGE and probed with a rabbit anti-rat NEP/CD10 polyclonal Ab. Sizes are shown in kDa, and the arrowhead indicates the band corresponding to NEP/CD10. The data shown are representative of three separate experiments. C, the expression of NEP/CD10 on thymocytes was evaluated by immunofluorescence and FACS analysis using a rabbit anti-rat CD10/NEP polyclonal Ab. Normal rabbit serum was used as a negative control, and goat FITC-conjugated anti-rabbit was used as the secondary Ab. The data are representative of three separate experiments. The gray area represents the relative cell number of NEP/CD10-positive cells. D, thymocytes were stained with FITC-conjugated anti-rat CD4, PE-conjugated anti-rat CD8 mAb, and rabbit anti-rat NEP/CD10 polyclonal Ab, followed by biotin GAR IgG and tricolor-conjugated streptavidin. The expression of CD4 and CD8 antigens was analyzed by flow cytometry on R1-gated CD10+ rat thymocytes. Data are representative of three separate experiments. Numbers in the cytogram represent the percentage of positive cells.

	Results

Top Abstract Materials and Methods Results Discussion References

 
Expression of CD10/NEP on Rat Thymocytes. Previous findings showed the presence of NEP/CD10 on unfractionated human thymocytes (Mari et al., 1994; Guérin et al., 1997), but direct evidence for the expression of CD10/NEP on distinct thymocyte subset is still lacking.

We initially evaluated the expression of NEP/CD10 mRNA on Ficoll gradient-purified (>99% CD5⁺) rat thymocytes and brain tissue used as positive control for the PCR analysis (Pollard et al., 1989). A PCR product of expected size was identified in both rat thymocytes and brain tissue (Fig. 1A).

Expression of NEP/CD10 was then assessed at the protein level. Western blot analysis of thymocyte lysates revealed a single band with an apparent molecular mass of 100 kDa probably corresponding to NEP/CD10, because a similar band was evident in lysates of rat brain tissue (Fig. 1B). No reactivity was observed with normal rabbit serum used as negative control (data not shown). Immunofluorescence and FACS analysis revealed that NEP/CD10 protein is expressed on CD5⁺ thymocytes (Fig. 1C), mainly on the CD4⁺CD8⁺ (DP) subset (Fig. 1D).

Thiorphan Administration Up-Regulates Both PPT-A mRNA and SP Protein Expression on Rat Thymocytes. Recent evidence indicates that SP modulates PPT-A mRNA and SP levels in an autocrine fashion upon interaction with its receptor NK₁R (Bae et al., 2002; Santoni et al., 2002). Thus, to assess whether inhibition of SP degradation by in vivo thiorphan administration could affect SP expression on CD5⁺ rat thymocytes, we determined both the expression of SP precursor PPT-A at the mRNA level and SP protein levels. As shown by quantitative RT-PCR analysis, a marked increase (approximately 2-fold) in PPT-A mRNA levels was observed on thymocytes from thiorphan-administered rats compared with untreated or vehicle-treated rats (Fig. 2A). No major differences were found by comparing PPT-A mRNA level in thymocytes from vehicle-administered versus untreated rats.

View larger version (14K): [in this window] [in a new window]   Fig. 2. Thiorphan up-regulates PPT-A mRNA and SP protein expression on thymocytes. A, PPT-A expression was evaluated by quantitative real-time PCR in triplicate on thymocytes (>99% CD5+ cells) from untreated, vehicle-, or thiorphan-administered rats. Results (mean ± S.D.) are normalized for β-actin expression. PPT-A levels were expressed as relative -fold with respect to the untreated rats used as controls. Data shown are representative of three separate experiments. **, p < 0.01 was determined by ANOVA, comparing thiorphan- or vehicle-administered rats with untreated rats. B, the expression of SP on thymocytes (>99% CD5+ cells) from untreated, vehicle-, thiorphan-, SR140333-, or thiorphan plus SR140333 antagonist-administered rats was evaluated by double immunofluorescence and FACS analysis using a mouse PE-conjugated anti-rat CD5 mAb and a goat anti-rat SP polyclonal Ab. Normal goat serum was used as a negative control, and FITC-conjugated rabbit anti-goat was used as the secondary Ab. The data are expressed as mean ± S.D. of three separate experiments. **, p < 0.01 was determined by ANOVA, comparing thiorphan-administered rats with untreated, vehicle-, SR140333-, or thiorphan plus SR140333-administered rats.

We also analyzed the mRNA levels of other peptide mediators that are NEP substrates, namely CGRP, ET-1, and TMPO, and we found that thiorphan treatment resulted in a significant increase of TMPO levels only (Fig. 3). Taken together, these findings suggest that SP de novo synthesis by thymocytes is profoundly susceptible to microenvironmental changes of SP levels induced by inhibition of NEP-mediated SP degradation.

View larger version (10K): [in this window] [in a new window]   Fig. 3. Thiorphan treatment enhances TMPO, but not CGRP, ET-1 mRNA expression on thymocytes. CGRP (A), ET-1 (B), and TMPO (C) mRNA expression was evaluated by quantitative real-time PCR in triplicate on thymocytes (>99% CD5+ cells) from untreated, vehicle-, or thiorphan-administered rats. Results (mean ± S.D.) are normalized for β-actin expression. CGRP, ET-1, and TMPO levels were expressed as relative -fold with respect to untreated rats used as controls. Data shown are representative of two separate experiments. **, p < 0.01 was determined by ANOVA, comparing thiorphan- or vehicle-administered rats with untreated rats.

Thiorphan Administration Increases NK₁R mRNA and Protein Expression on Rat Thymocytes. Exogenous SP has been reported to up-regulate NK₁R mRNA expression in vitro in a ligand-dependent manner. Thus, we analyzed whether thiorphan administration could modulate NK₁R expression on thymocytes at mRNA and protein levels.

RT-PCR analysis on total RNA of thymocytes from thiorphan-administered rats showed a 4-fold increase in NK₁R mRNA levels, whereas no significant differences were observed on thymocytes from vehicle-administered versus untreated rats (Fig. 4A). We further evaluated the expression of NK₁R by immunofluorescence and FACS analysis on CD5⁺ thymocytes from thiorphan-administered rats treated or not with SR140333; thiorphan administration induced a 3-fold increase of NK₁R expression, which was completely inhibited by SR140333 (Fig. 4B). Overall, these results indicate that NK₁R thymocytes expression is up-regulated after its activation by endogenous SP.

View larger version (14K): [in this window] [in a new window]   Fig. 4. Thiorphan up-regulates NK1R mRNA and protein expression on rat thymocytes. A, NK1R mRNA expression was evaluated by quantitative real-time PCR in triplicate on thymocytes (>99% CD5+ cells) from untreated, vehicle-, or thiorphan-administered rats. Results (mean ± S.D.) are normalized for β-actin expression. NK1R levels were expressed as relative -fold with respect to untreated rats used as controls. Data shown are representative of three separate experiments. **, p < 0.01 was determined by ANOVA, comparing thiorphan- or vehicle-administered rats with untreated rats. B, the expression of NK1R protein on thymocytes (>99% CD5+ cells) from untreated, vehicle-, thiorphan-, SR140333-, or thiorphan plus SR140333 antagonist-administered rats was evaluated by double immunofluorescence and FACS analysis using a mouse PE-conjugated anti-rat CD5 mAb and a goat anti-rat NK1R polyclonal Ab. Normal goat serum was used as a negative control, and FITC-conjugated rabbit anti-goat was used as the secondary Ab. The data are expressed as mean ± S.D. of three separate experiments. **, p < 0.01 was determined by ANOVA, comparing thiorphan-administered rats with untreated, vehicle-, SR140333-, or thiorphan plus SR140333-administered rats.

Effect of Thiorphan Administration on Total CD10⁺ Thymocyte Number and Subset Distribution. Exogenous SP administration has been reported to increase thymocyte numbers (Santoni et al., 2002). Thus, to investigate the functional role of endogenous SP on thymocytes expressing the NEP/CD10 molecule, we initially investigated the ability of thiorphan to affect thymocyte number and subset distribution. NEP/CD10 is expressed on 45.5% of total thymocytes on both DP and CD4⁺ cells. We found that thiorphan enhances total thymocyte number, and this increase was completely reversed by simultaneous SR140333 administration (Table 1). Cytofluorimetric analysis also showed that thiorphan administration markedly increased the percentage and absolute number of CD10⁺CD4⁺ and CD10⁺ DP thymocytes; in contrast, SR140333 that was administered simultaneously with thiorphan reverted the thiorphan-induced stimulatory effect (Table 1).

Thiorphan Administration Induces IL-2 Production, IL-2 Receptor (CD25) Chain Expression, and Stimulates ConA-Induced Thymocyte Proliferation. We have previously demonstrated that exogenous SP administration induces CD25 expression, IL-2 production, and stimulates ConA-induced thymocyte proliferation (Santoni et al., 2002). Thus, to investigate the involvement of the IL-2/IL-2 receptor (IL-2R) pathway in the endogenous SP-induced stimulation of thymocyte proliferation, we analyzed the ability of in vivo thiorphan administration to modulate IL-2 and CD25 expression. By ELISA assay, we found that IL-2 is already present in the culture supernatants of unstimulated thymocytes from thiorphan-administered rats and further increases in response to ConA (Fig. 5A). In addition, SR140333 that was simultaneously administered with thiorphan inhibited the endogenous SP-induced IL-2 production; moreover, FACS analysis revealed that thiorphan administration enhances (from 10 to 68%), and SR140333 markedly reduces (from 68 to 24%), basal CD25 expression (Fig. 5B).

View larger version (17K): [in this window] [in a new window]   Fig. 5. Thiorphan induces IL-2 release, IL-2R chain expression, and stimulates ConA-induced rat thymocyte proliferation. A, supernatants from 24-h cultures of unstimulated or ConA-stimulated thymocytes from untreated, vehicle-, thiorphan, SR140333-, or thiorphan plus SR140333-administered rats were tested for the presence of IL-2 by ELISA. The data are expressed as mean ± S.D. of three separate experiments performed in triplicate. **, p < 0.01 was determined by ANOVA, comparing thiorphan-administered rats with untreated, vehicle-, SR140333-, or thiorphan plus SR140333-administered rats and SR140333 with untreated or vehicle-administered rats. B, thymocytes from untreated, vehicle-, thiorphan, SR140333-, or thiorphan plus SR140333-administered rats were stained with mouse FITC-conjugated anti-rat CD5 and PE-conjugated anti-rat CD25 mAbs and tested by two-color immunofluorescence and FACS analysis. The data are expressed as mean ± S.D. of three separate experiments. Statistical analysis was determined by ANOVA; **, p < 0.01 as described in A. C, thymocytes from untreated, vehicle-, thiorphan, SR140333-, or thiorphan plus SR140333-administered rats were tested for their ability to proliferate in response to ConA (5.0 µg/ml). Thymocyte proliferation was evaluated at 72 h by [3H]TdR incorporation and was expressed as stimulation index. The data are represented as mean ± S.D. of three separate experiments performed in triplicate. Statistical analysis was determined by ANOVA; **, p < 0.01 as described in A.

Thiorphan Administration Rescues Rat Thymocytes from Spontaneous and NK₁R Antagonist-Induced Apoptosis. We have previously demonstrated that exogenous SP administration rescues thymocytes from spontaneous and NK₁R antagonist-induced apoptosis (Santoni et al., 2002). Thus, we investigated, by biparametric flow cytometric analysis, whether increased levels of endogenous SP by means of thiorphan administration could also act as a survival factor by rescuing thymocytes from spontaneous apoptosis.

Thiorphan administration significantly increased the percentage (from 78 to 91) of PI^-/AnnV^- intact thymocytes. These effects were reversed by SR140333 administration, as evidenced by the reduced percentage of both PI^-/AnnV⁺ early and PI⁺/AnnV⁺ late apoptotic or necrotic thymocytes; moreover, as reported previously (Santoni et al., 2002), SR140333 induced apoptosis as shown by the increased percentage (from 16 to 33%) of PI^-/AnnV⁺ apoptotic thymocytes (Table 2). Overall, these data demonstrate the capacity of thiorphan to protect thymocytes from both spontaneous and NK₁R antagonist-induced apoptosis through increased levels of endogenous SP.

Thiorphan Administration Induces Akt Phosphorylation on Rat Thymocytes. To investigate the signaling pathway underlying the SP-mediated thymocyte rescue from apoptosis, we examined whether thiorphan administration could result in changes of Akt phosphorylation, a signaling event controlling cell survival (Coffer et al., 1998). Western blotting using an antibody to Ser473-phosphospecific Akt revealed increased (3-fold) Akt phosphorylation on rat thymocytes upon thiorphan administration (Fig. 6). In contrast, the SR140333 administered simultaneously with thiorphan markedly inhibited thiorphan-induced Akt phosphorylation. Like the NEP inhibitor, SP administration stimulated Akt phosphorylation, further supporting the fact that thiorphan-induced Akt phosphorylation is mediated by increased levels of endogenous SP.

View larger version (28K): [in this window] [in a new window]   Fig. 6. Thiorphan induces Ser473 Akt phosphorylation on rat thymocytes. Lysates of thymocytes from untreated, vehicle-, thiorphan-, SR140333-, thiorphan plus SR140333-, or SP-administered rats, resuspended in serum-free media, were analyzed for Ser473-phosphorylated Akt by Western blotting using a rabbit anti-rat phospho Akt Ab. The blot was stripped and reprobed with rabbit anti-rat Akt polyclonal Ab. The relative Akt Ser473 phosphorylation levels on thymocytes were determined by densitometric analysis using a ChemiDoc apparatus and Akt protein level as a loading control. Data are represented as mean + SD of three separate experiments. Statistical analysis was determined by ANOVA; **, p < 0.01 as described above.

View larger version (14K): [in this window] [in a new window]   Fig. 7. Thiorphan modulates survivin and procaspase-3 mRNA expression in rat thymocytes. Survivin (A) and procaspase-3 (B) mRNA level were evaluated by quantitative real-time PCR on thymocytes (>99% CD5+ cells) from untreated, vehicle-, thiorphan-, SR140333-, thiorphan plus SR140333-, or SP-administered rats. Results (mean ± S.D.) are normalized for β-actin expression. Survivin and procaspase-3 levels were expressed as relative fold with respect to untreated rats used as controls. Data shown are representative of three separate experiments. **, p < 0.01 was determined by ANOVA, comparing thiorphan-administered rats with untreated, vehicle-, SR140333-, or thiorphan plus SR140333-administered rats and SP with untreated, vehicle-, SR140333-, or thiorphan-administered rats.

Thiorphan Administration Reverts Caspase-3 Activation on Rat Thymocytes. Constitutive caspase-3 activity has been described in mouse DP thymocytes (Jiang et al., 1999), and recently the ability of survivin to interact with, and to inhibit, caspase-3 activity has been demonstrated (Ohashi et al., 2004). Thus, we analyzed the state of caspase-3 activation in rat thymocytes from untreated, vehicle-, thiorphan-, SR140333, thiorphan plus SR140333-, or SP-treated rats. We found that caspase-3 is constitutively activated in untreated thymocytes, as demonstrated by the appearance of the 17-kDa fragment of caspase-3 (Fig. 8). Thiorphan administration significantly inhibited caspase-3 activation in an NK₁R-dependent manner, as shown by the ability of SR140333 administration to completely revert the thiorphan-mediated effects. Likewise, exogenous SP inhibited the basal level of caspase-3 activation. No major differences on caspase-3 activation were observed in thymocytes from vehicle-treated versus untreated rats. Overall, these results suggest that thiorphan in vivo induces SP-dependent and NK₁R-mediated inhibition of caspase-3 activation on rat thymocytes.

View larger version (34K): [in this window] [in a new window]   Fig. 8. Thiorphan administration inhibits caspase-3 activation in rat thymocytes. Lysates of thymocytes from untreated, vehicle-, thiorphan-, SR140333-, thiorphan plus SR140333-, or SP-administered rats were separated on SDS-PAGE and probed with rabbit anti-caspase-3 polyclonal Ab. Sizes are shown in kDa, and arrows indicate the bands corresponding to the procaspase-3 and its caspase-3 17-kDa cleaved fragment. The relative active caspase-3 levels were determined by densitometric analysis using a ChemiDoc apparatus and -tubulin level as loading control. Data are represented as mean ± S.D. of three separate experiments. Statistical analysis was determined by ANOVA; **, p < 0.01 as described above.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
The local bioactivity of SP in the rat thymus seems to be tightly controlled by its synthesis/release from sensory nerves (Jurjus et al., 1998) and thymocytes (Santoni et al., 2002), the presence of NK₁R on distinct thymocyte subsets (Santoni et al., 2002), and the local expression of SP-degrading enzymes such as NEP/CD10 (Mari et al., 1994; Guérin et al., 1997).

In this study, we provide evidence for the expression of NEP/CD10 by rat thymocytes and its involvement in the regulation of thymocyte proliferation and survival. NEP/CD10 expression was detected on approximately 45% of highly purified CD5⁺ thymocytes, on both DP and CD4⁺ subsets. The overlapping expression of NK₁R and NEP/CD10 on DP thymocytes suggests a functional role of NEP/CD10 in the SP-mediated control of thymus homeostasis and T cell development (Santoni et al., 1999, 2002). Our findings are consistent with a previous report by Okamoto et al. (1994), demonstrating that coexpression of NEP/CD10 and NK₁Rin rat kidney transfected cells is associated with reduced SP-mediated effects due to increased SP breakdown.

Peptide signaling molecules are controlled at both synthesis/release and degradation synthesis/release levels. We have previously reported the expression of mRNA for the SP precursor, PPT-A, and SP in CD5⁺ thymocytes, and their ability to spontaneously release SP (Santoni et al., 2002).

In this study, we demonstrate that thiorphan administration, by reducing SP degradation, markedly enhances the local bioavailability, and consequently the NK₁R-mediated SP biological activities on thymocytes are magnified. Indeed, we found that simultaneous administration of thiorphan and the NK₁R antagonist SR140333 resulted in down-regulated expression of PPT-A mRNA and SP protein as well as of NK₁R mRNA and protein levels. These findings are consistent with previous evidence indicating that SP itself promotes its own synthesis and NK₁R expression in an NK₁R-dependent manner (Bae et al., 2002; Santoni et al., 2002). In addition, we also observed that thiorphan administration enhanced the thymocyte mRNA levels of TMPO, but not those of other thymic NEP substrates including ET-1 and CGRP.

Our study also provides evidence that inhibition of NEP/CD10-mediated SP breakdown on rat thymocytes results in enhanced SP-mediated functional responses. A marked increase of total CD10 thymocyte numbers was detected in thiorphan-administered rats, and this increase was specifically inhibited by the SR140333 antagonist. The increased thymocyte number was accompanied by enhanced IL-2 production, increased CD25 expression, and ConA-induced thymocyte proliferation, suggesting that the IL-2/IL-2R system plays a major role in the NEP inhibitor-induced SP-mediated regulation of thymocyte-proliferative response.

The evidence that the NK₁R antagonist did not completely reverse thiorphan-mediated increase of CD25 expression and IL-2 release, together with the observation that thiorphan administration enhances TMPO mRNA expression, also suggest the involvement of TMPO NEP substrate in the control of these functional responses. In this regard, TMPO has been described to stimulate IL-2 release in T cells (Guérin et al., 1997).

In addition, in accordance with previous evidence showing that NEP/CD10 regulates cell survival (Shipp and Look, 1993; Sumitomo et al., 2001), and that exogenous SP protects thymocytes from spontaneous and NK₁R antagonist-induced apoptosis (Santoni et al., 2002), we found that CD10/NEP activity regulates the expression and activation of an important component of antiapoptotic responses, namely the Akt/survivin pathway, by controlling SP levels.

Akt activation is a critical signaling event that controls survival of a variety of cell types, including thymocytes (Jones et al., 2000; Kelly et al., 2002), and overexpression of activated Akt was reported to selectively regulate survival of murine DP and SP thymocytes (Jones et al., 2000). In addition, a recent report describes the ability of SP to stimulate Akt phosphorylation and antiapoptotic responses on human colonocytes (Koon et al., 2007).

In this regard, we found that thiorphan administration increased Ser473 Akt phosphorylation on rat thymocytes in an NK₁R-dependent manner, and that coadministration of SR140333 antagonist and thiorphan completely reverted the NEP-mediated SP stimulation of Akt phosphorylation; moreover, we observed that administration of SP itself stimulates Akt phosphorylation.

We also showed that thymocytes from thiorphan- or SP-treated rats exhibited increased mRNA expression of survivin, an antiapoptotic inhibitor involved in the Akt promotion of cell survival (Ambrosini et al., 1998; Dan et al., 2004). The importance of survivin in the regulation of thymocyte survival is also supported by recent evidence describing the fact that loss of survivin blocks the transition from double-negative to DP thymocytes and triggers their growth arrest and cell death (Okada et al., 2004). Like other thiorphan-mediated effects, enhanced survivin mRNA expression was also attributable to increased SP bioavailability and activity because it was reverted by simultaneous administration of SR140333.

The antiapoptotic activity of survivin can be mediated by its ability to interact with caspase-3 and to inhibit caspase-3-mediated apoptosis (Ohashi et al., 2004). In this regard, in agreement with previous findings on mouse DP thymocytes (Jiang et al., 1999), we observed that caspase-3 is constitutively activated in untreated thymocytes; thiorphan administration completely inhibited the caspase-3 activation and parallely increased the basal level of procaspase-3 protein, and this inhibition was reverted by SR140333, thus supporting a role for SP in thiorphan-mediated effects.

Our findings provide the first evidence for a critical in vivo role of NEP/CD10 in the regulation of thymocyte proliferation and survival by controlling the endogenous levels of SP. In addition, they shed light on some of the antiapoptotic and proapoptotic molecular mechanisms, namely activation of the PI3-K/Akt/survivin pathway and inhibition of procaspase-3 expression and caspase-3 activity, underlying SP-mediated promotion of thymocyte survival. Our results are consistent with previous evidence indicating that PI3-K inhibitors down-regulate survivin expression and increase the caspase activity that is associated with cell death (Sommer et al., 2003).

At present, it is unclear whether the in vivo antiapoptotic effects mediated by increased bioavailability of endogenous SP or by exogenous SP administration are directly and/or indirectly related to its ability to stimulate IL-2 release. In this regard, growing evidence indicates that SP exerts direct antiapoptotic effects, and they have also been attributed to its ability to trigger Akt activation (Koon et al., 2007). On the other hand, an important role for IL-2 in regulating T cell apoptosis and survival, activating the PI3-K/Akt pathway (Jones et al., 2000; Kelly et al., 2002), and up-regulating survivin expression has been also reported. Nonetheless, unlike SP, IL-2 has not been implicated in modulation of procaspase-3 expression and/or caspase-3 activity (Sabbagh et al., 2004). This finding, together with the ability of exogenous SP to stimulate Akt phosphorylation and survivin expression, strongly supports the direct role of SP in mediating antiapoptotic responses in rat thymocytes.

What is the pathophysiological role of the SP/NK₁R/NEP pathway? The expression of SP, NK₁R, and NEP/CD10 on thymocyte subsets strongly supports the hypothesis that continuous SP release in the thymus regulates T cell homeostasis and development by acting as both survival and growth-promoting factors. By controlling the availability of SP in the thymic environment under basal circumstances, NEP/CD10 represents an important negative feedback mechanism that limits SP-immunostimulatory effects. Moreover, increased SP levels have been associated with immune-mediated inflammatory conditions such as arthritis, asthma, allergic contact dermatitis, and inflammatory bowel diseases (Brain and Cox, 2006), and depletion of SP levels by NEP/CD10 has been found to ameliorate these inflammatory responses. A better understanding of the mechanisms by which NEP/CD10 regulates SP bioavailability and function in the thymus will provide novel insights for potential NEP/CD10-based therapeutic intervention in the treatment of these inflammatory and autoimmune disorders.

	Acknowledgements

 
We thank Cucculelli Marino for technical assistance and Angela Santoni for critical reading of the manuscript.

	Footnotes

 
This work was supported in part by the University of Camerino.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.108.138719.

ABBREVIATIONS: NEP, neutral endopeptidase; ET-1, endothelin-1; TMPO, thymopoietin; SP, substance P; CGRP, calcitonin gene-related peptide; NK₁R, neurokinin-1 receptor; PPT-A, preprotachykinin A; DP, double positive; SR140333, (S)1-{2-[3(3-4-dichlorophenyl)-1-(3-isopropoxyphenylacetyl)piperidine-3-yl]ethyl}-4-pheny-1-azoniabicyclo[2.2.2]octane, chloride; PI3-K, phosphatidylinositol-3 kinase; IAP, inhibitor of apoptosis; FACS, fluorescent-activated cell sorting; mAbs, monoclonal antibodies; PE, phycoerythrin; FITC, fluorescein isothiocyanate; RAG, rabbit anti-goat; GAR, goat anti-rabbit; HRP, horseradish peroxidase; Ab, antibody; PAGE, polyacrylamide gel electrophoresis; ConA, concanavalin A; [³H]TdR, tritiated thymidine; IL, interleukin; ELISA, enzyme-linked immunosorbent assay; AnnV, annexin V; PI, propidium iodine; PCR, polymerase chain reaction; ANOVA, analysis of variance; RT, real time; IL-2R, IL-2 receptor.

Address correspondence to: Dr. Giorgio Santoni, Department of Experimental Medicine and Public Health, University of Camerino, Via Madonna delle Carceri 9, 62032, Camerino, Italy. E-mail: giorgio.santoni{at}unicam.it

	References

Top Abstract Materials and Methods Results Discussion References

 

Ambrosini G, Adida C, Sirugo G, and Altieri DC (1998) Induction of apoptosis and inhibition of cell proliferation by survivin gene targeting. J Biol Chem 273: 11177-11182.[Abstract/Free Full Text]
Bae SJ, Matsunaga Y, Takenaka M, Tanaka Y, Hamazaki Y, Shimizu K, and Katayama I (2002) Substance P induced preprotachykinin-A mRNA, neutral endopeptidase mRNA and substance P in cultured normal fibroblasts. Int Arch Allergy Immunol 127: 316-321.[CrossRef][Medline]
Blalock JE and Smith EM (1985) The immune system: our mobile brain? Immunol Today 6: 115-117.
Brain SD and Cox HM (2006) Neuropeptides and their receptors: innovative science providing novel therapeutic targets. Br J Pharmacol 147 (Suppl 1): S202-S211.[CrossRef][Medline]
Coffer PJ, Jin J, and Woodgett JR (1998) Protein kinase B (c-Akt): a multifunctional mediator of phosphatidylinositol 3-kinase activation. Biochem J 335: 1-13.[Medline]
Dan HC, Jiang K, Coppols D, Hamilton A, Nicosia SV, Sebti SM, and Cheng JQ (2004) Phosphatidylinositol-3-OH kinase/Akt and survivin pathways as critical targets for geranylgeranyltransferase I inhibitor-induced apoptosis. Oncogene 23: 706-715.[CrossRef][Medline]
Edmonds-Alt X, Doutremepuich JD, Heaulme M, Neliat G, Santucci V, Steinberg R, Vilain P, Bichon D, Ducoux JP, Proietto V, et al. (1993) In vitro and in vivo biological activities of SR140333, a novel potent non-peptide tachykinin NK1 receptor antagonist. Eur J Pharmacol 250: 403-413.[CrossRef][Medline]
Grady E, Yoshimi SK, Maa J, Valeroso D, Vartanian RK, Rahim S, Kim EH, Gerard C, Gerard N, Bunnett NW, et al. (2000) Substance P mediates inflammatory oedema in acute pancreatitis via activation of the neurokinin-1 receptor in rats and mice. Br J Pharmacol 130: 505-512.[CrossRef][Medline]
Guérin S, Mari B, Maulon L, Belhacène N, Marguet D, and Auberger P (1997) CD10 plays a specific role in early thymic development. FASEB J 11: 376-381.[Abstract]
Institute of Laboratory Animal Resources (1996) Guide for the Care and Use of Laboratory Animals 7th ed. Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council, Washington DC.
Jiang D, Zheng L, and Lenardo MJ (1999) Caspases in T-cell receptor-induced thymocyte apoptosis. Cell Death Differ 6: 402-411.[CrossRef][Medline]
Jones RG, Parsons M, Bonnard M, Chan VS, Yeh WC, Woodgett JR, and Ohashi PS (2000) Protein kinase B regulates T lymphocyte survival, nuclear factor kB activation, and Bcl-X_L levels in vivo. J Exp Med 191: 1721-1733.[Abstract/Free Full Text]
Jurjus AR, More N, and Walsh RJ (1998) Distribution of substance P positive cells and nerve fibers in the rat thymus. J Neuroimmunol 90: 143-148.[CrossRef][Medline]
Keeble J, Blades M, Pitzalis C, Castro da Rocha FA, and Brain SD (2005) The role of substance P in microvascular responses in murine joint inflammation. Br J Pharmacol 144: 1059-1066.[CrossRef][Medline]
Kelly E, Won A, Refaeli Y, and Van Parijs L (2002) IL-2 and related cytokines can promote T cell survival by activating Akt. J Immunol 168: 597-603.[Abstract/Free Full Text]
Kerr MA and Kenny AJ (1974) The purification and specificity of a neutral endopeptidase from rabbit kidney brush border. Biochem J 137: 477-488.[Medline]
Koon H-W, Zhao D, Zhan Y, Moyer MP, and Pothoulakis C (2007) Substance P mediates antiapoptotic responses in human colonocytes by Akt activation. Proc Natl Acad Sci U S A 104: 2013-2018.[Abstract/Free Full Text]
Lu B, Gerard NP, Kolakowski LF Jr, Bozza M, Zurakowski D, Finco O, Carroll MC, and Gerard C (1995) Neutral endopeptidase modulation of septic shock. J Exp Med 181: 2271-2275.[Abstract/Free Full Text]
Maa J, Grady EF, Kim EH, Yoshimi SK, Hutter MM, Bunnett NW, and Kirkwood KS (2000) NK-1 receptor desensitization and neutral endopeptidase terminate SP-induced pancreatic plasma extravasation. Am J Physiol Gastrointest Liver Physiol 279: G726-G732.[Abstract/Free Full Text]
Maggi CA (1995) The mammalian tackykinin receptors. Gen Pharmacol 26: 911-944.[Medline]
Mari B, Breittmayer JP, Guerin S, Belhacene N, Peyron JF, Deckert M, Rossi B, and Auberger P (1994) High levels of functional endopeptidase 24.11 (CD10) activity on human thymocytes: preferential expression on immature subsets. Immunology 82: 433-438.[Medline]
Matsas R, Kenny AJ, and Turner AJ (1984) The metabolism of neuropeptides. The hydrolysis of peptides including enkephalins, tackykinins and their analogues, by endopeptidase-24.11. Biochem J 223: 433-440.[Medline]
Michalski C, Autschbach F, Selvaggi F, Shi X, Di Moia FF, Regge A, Müller MW, Di Sebastiano P, Büchler MW, Giese T, et al. (2007) Increase in substance P precursor mRNA in non inflame small-bowel sections in patients with Cronn's disease. Am J Surg 193: 476-481.[CrossRef][Medline]
Naquet P and Pierres M (1991) Cell-surface enzymes and lymphocyte functions. Curr Opin Immunol 3: 326-329.[CrossRef][Medline]
Ohashi H, Tagaki H, Oh H, Suzuma K, Suzuma I, Miyamoto N, Uemura A, Watanabe D, Murakami T, Sugaya T, et al. (2004) Phosphatidylinositol 3-kinase/Akt regulates angiotensin II-induced inhibition of apoptosis in microvascular endothelial cells by governing survivin expression and suppression of caspase-3 activity. Circulation Res 94: 785-793.[Abstract/Free Full Text]
Okada H, Bakal C, Shahinian A, Elia A, Wakeham A, Suh WK, Duncan GS, Ciofani M, Rottapel R, Zúñiga-Pflücker JC, et al. (2004) Survivin loss in thymocytes triggers p53-mediated growth arrest and p53-independent cell death. J Exp Med 199: 399-410.[Abstract/Free Full Text]
Okamoto A, Lovett M, Payan DG, and Bunnett NW (1994) Interaction between neutral peptidase (EC 3.4.24.11) and the substance P (NK-1) receptor expressed in mammalian cells. Biochem J 299: 683-693.[Medline]
Pollard H, Bouthenet ML, Moreau J, Souil E, Verroust P, Ronco P, and Schwartz JC (1989) Detailed immunoautoradiographic mapping of enkephalinase (EC 3.4.24.11) in rat central nervous system: comparison with enkephalins and substance P. Neuroscience 30: 339-376.[CrossRef][Medline]
Qian BF, Zhou GQ, Hammarström ML, and Danielsson A (2001) Both substance P and its receptor are expressed in mouse intestinal T lymphocytes. Neuroendocrinology 73: 358-368.[CrossRef][Medline]
Ritz J, Nadler LM, Bhan AK, Notis-McConarty J, Pesando JM, and Schlossman SF (1981) Expression of a common acute lymphoblastic leukemia antigen (CALLA) by lymphomas of B-cell and T-cell lineage. Blood 58: 648-652.[Abstract/Free Full Text]
Roques BP, Fournié-Zaluski MC, Soroca E, Lecomte JM, Malfroy B, Llorens C, and Schwartz JC (1980) The enkephalinase inhibitor thiorphan shows antinociceptive activity in mice. Nature 288: 286-288.[CrossRef][Medline]
Sabbagh L, Kaech SM, Bourbonnière M, Woo M, Cohen LY, Haddad EK, Labrecque N, Ahmed R, and Sékaly RP (2004) The selective increase in caspase-3 expression in effector but not memory T cells allows susceptibility to apoptosis. J Immunol 173: 5425-5433.[Abstract/Free Full Text]
Santoni G, Perfumi MC, Spreghini E, Romagnoli S, and Piccoli M (1999) Neurokinin type-1 receptor antagonist inhibits enhancement of T cell functions by substance P in normal and neuromanipulated capsaicin-treated rats. J Neuroimmunol 93: 15-25.[CrossRef][Medline]
Santoni G, Amantini C, Lucciarini R, Pompei P, Perfumi M, Nabissi M, Morrone S, and Piccoli M (2002) Expression of substance P and its neurokinin-1 receptor on thymocytes: functional relevance in the regulation of thymocyte apoptosis and proliferation. Neuroimmunomodulation 10: 232-246.[CrossRef][Medline]
Scholzen TE, Steinhoff M, Bonaccorsi P, Klein R, Amadesi S, Geppetti P, Lu B, Gerard NP, Olerud JE, Luger TA, et al. (2001) Neutral endopeptidase terminates substance P-induced inflammation in allergic contact dermatitis. J Immunol 166: 1285-1291.[Abstract/Free Full Text]
Shipp MA and Look AT (1993) Hematopoietic differentiation antigens that are membrane-associated enzymes: cutting is the key! Blood 82: 1052-1070.[Free Full Text]
Sommer KW, Schamberger CJ, Schmidt GE, Sasgary S, and Cerni C (2003) Inhibitor of apoptosis protein (IAP) survivin is upregulated by oncogenic c-H-Ras. Oncogene 22: 4266-4280.[CrossRef][Medline]
Sumitomo M, Milowsky MI, Shen R, Navarro D, Dai J, Asano T, Hayakawa M, and Nanus DM (2001) Neutral endopeptidase inhibits neuropeptide-mediated transactivation of the insulin-like growth factor receptor-Akt cell survival pathway. Cancer Res 61: 3294-3298.[Abstract/Free Full Text]
Turner AJ and Tanzawa K (1997) Mammalian membrane metallopeptidases: NEP, ECE, KELL, and PEX. FASEB J 11: 355-364.[Abstract]
Yang ZZ, Tschopp O, Baudry A, Dümmler B, Hynx D, and Hemmings BA (2004) Physiological functions of protein kinase B/Akt. Biochem Soc Trans 32: 350-354.[CrossRef][Medline]

This article has been cited by other articles:

				J. Meshki, S. D. Douglas, J.-P. Lai, L. Schwartz, L. E. Kilpatrick, and F. Tuluc Neurokinin 1 Receptor Mediates Membrane Blebbing in HEK293 Cells through a Rho/Rho-associated Coiled-coil Kinase-dependent Mechanism J. Biol. Chem., April 3, 2009; 284(14): 9280 - 9289. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.108.138719v1 327/1/215    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Amantini, C. Articles by Santoni, G. Search for Related Content PubMed PubMed Citation Articles by Amantini, C. Articles by Santoni, G.