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Neuropeptide S stimulates human monocyte chemotaxis via NPS receptor activation
Peptides 39 (2013) 16–20
Contents lists available at SciVerse ScienceDirect
Peptides journal homepage: www.elsevier.com/locate/peptides
Neuropeptide S stimulates human monocyte chemotaxis via NPS receptor activation M. Filaferro a , C. Novi a , V. Ruggieri a , S. Genedani a , S. Alboni a , D. Malagoli b , G. Caló c , R. Guerrini d , G. Vitale a,∗ a
Department of Biomedical Sciences, Section of Pharmacology, University of Modena and Reggio Emilia, Via G. Campi 287, 41125 Modena, Italy Department of Biology, University of Modena and Reggio Emilia, Via G. Campi, 213/d, 41125 Modena, Italy c Department of Experimental and Clinical Medicine, Section of Pharmacology and Neuroscience Center and National Institute of Neuroscience, University of Ferrara, Via Fossato di Mortara 19, 44121 Ferrara, Italy d Department of Pharmaceutical Sciences and LTTA, University of Ferrara, Via Fossato di Mortara 19, 44100 Ferrara, Italy b
a r t i c l e
i n f o
Article history: Received 19 June 2012 Received in revised form 30 October 2012 Accepted 31 October 2012 Available online 8 November 2012 Keywords: Neuropeptide S fMLP Human NPS receptor Monocytes Chemotaxis NPSR antagonists
a b s t r a c t Neuropeptide S (NPS) produces several biological actions by activating a formerly orphan GPCR, now named NPS receptor (NPSR). It has been previously demonstrated that NPS stimulates murine leukocyte chemotaxis in vitro. In the present study we investigated the ability of NPS, in comparison with the proinflammatory peptide formyl-Met-Leu-Phe (fMLP), to stimulate human monocyte chemotaxis. At a concentration of 10−8 M fMLP significantly stimulated chemotaxis. NPS produced a concentration dependent chemotactic action over the concentration range 10−12 to 10−5 M. The NPSR antagonists [dCys(t Bu)5 ]NPS, [t Bu-d-Gly5 ]NPS and SHA 68 were used to pharmacologically characterize NPS action. Monocyte chemoattractant effect of NPS, but not fMLP, was completely blocked by either peptide antagonists or SHA with the nonpeptide molecule being more potent. None of the NPSR antagonists modified per se random cell migration. Thus, the present study demonstrated that NPS is able to stimulate human monocyte chemotaxis and that this effect is entirely due to selective NPSR activation. © 2012 Elsevier Inc. All rights reserved.
1. Introduction Neuropeptide S (NPS) is expressed in specific brain regions and has various effects on the central nervous system. There is strong evidence that NPS promotes anxiolytic-like effects in rodents [22,30,33]. In addition, NPS induces hyperlocomotion [33], increases wakefulness and suppresses all stages of sleep [33], suppresses food intake [6,8,26] and facilitates extinction of conditioned fear [13]. A bioinformatic analysis of current genome databases revealed that the NPS peptide precursor gene is present in all vertebrates, including rodents and pigs [32,34] with the exception of fish [21]. NPS activates its G protein-coupled receptor (NPSR) at low nanomolar concentrations and induces elevation of intracellular Ca2+ and adenosine 3 ,5 -cyclic monophosphate levels. Various findings suggest that the NPS–NPSR system may play a role in modulating innate immunity and chronic inflammatory diseases particularly of epithelial barriers. In fact, NPS and NPSR mRNAs are co-localized in the human bronchial and colon epithelia suggesting
Abbreviations: NPS, neuropeptide S; NPSR, NPS receptor; fMLP, formyl-Met-LeuPhe. ∗ Corresponding author. Tel.: +39 059 2055370; fax: +39 059 2055376. E-mail address: [email protected] (G. Vitale). 0196-9781/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.peptides.2012.10.013
that NPSR may be activated by autocrine or paracrine mechanisms [19,29]. Leukocyte migration into tissues represents a key process in the pathogenesis of inflammatory diseases. Data obtained in clinical trials have convincingly shown that inhibition of leukocyte migration into target organs represents an effective therapeutic approach for diseases in which inflammation has a noxious effect [23]. Interestingly, in vitro studies showed that NPS is able to modulate macrophage phagocytosis; indeed, prestimulation of mouse macrophage cell line RAW 264.7 with NPS resulted in a dose-dependent increase in phagocytosis of fluoresceinlabeled Escherichia coli. Furthermore, stimulation with NPS (1 M) increased cell migration and decreased cell adhesion, suggesting that NPS is a chemotactic agent to murine macrophages in vitro [19]. Studies related to pig immune system showed the presence of NPSR in immune tissues, including thymus, spleen, jejunal lymph nodes and soft palatine tonsils [34] and NPS was found to act as a modulator of lymphocyte proliferation. Moreover, evidence from human genetic studies demonstrated the association of NPSR gene polymorphisms with chronic inflammatory diseases of the respiratory [14] and gastrointestinal [7] system. NPS and its receptor are thought to have a role in asthma pathogenesis; single nucleotide polymorphisms within NPSR have been shown to be associated with an increased prevalence of asthma [2]. Peripheral tissues and
M. Filaferro et al. / Peptides 39 (2013) 16–20
epithelia of several organs express NPSR isoforms A and B, including the intestinal mucosa and the skin, and NPSR appears to be upregulated in inflammation [27]. Indeed, NPS increased the proliferation of splenic lymphocytes and enhanced pulmonary alveolar macrophage function. Although NPS alone did not show a relevant effect on the production of IL-1, IL-6 and TNF-␣, NPS could significantly enhance their production induced by LPS [35]. Thus, it seems that NPS was able to modulate the inflammatory response only in the presence of exogenous antigens such as LPS [35]. Chemotaxis is a fundamental process in which cells migrate directionally when they are exposed to external specific chemical gradients. It is exhibited by a wide variety of cells types and involves distinct strategies that depend on the cell and the environmental conditions. Several members of the neuropeptide family exert chemotactic actions on blood monocytes. Furthermore, the immune system influences the central nervous system. At a molecular level, neuro and immune signal molecules (hormones, neurotransmitters, neuropeptides, and cytokines) and their receptors are indeed members of the same superfamilies which enable the mutual neuroimmune communication [31]. In the present study, we investigated the ability of NPS to influence human monocyte chemotaxis, in comparison with the proinflammatory peptide formyl-Met-Leu-Phe (fMLP). Moreover, the NPSR peptide antagonists [d-Cys(t Bu)5 ]NPS [4], [t Bu-dGly5 ]NPS [24], and nonpeptide antagonist SHA 68 [16,25] were used in order to pharmacologically characterize the effect of NPS. 2. Materials and methods 2.1. Preparation of human monocytes Human mononuclear cells were separated by LympholyteH (Cederlane Burlington, NC, USA) gradient centrifugation of peripheral blood (collected into heparinated Vacutainers) obtained from healthy volunteers. After centrifugation (20 min at 800 × g), monocytes were removed from the interface between plasma and Lympholyte-H. Mononuclear cells were washed twice in PBS, resuspended in RPMI 1640 medium (EuroClone Milano, Italy) supplemented with 0.1% BSA (SERVA Electrophoresis, Heidelberg, Germany), and diluted to a final concentration of 1.5 × 106 monocytes/ml. 2.2. Monocyte migration assay Migration assays were performed by using 48-well microchemotaxis chambers (Boyden modified, Neuroprobe, Gaithersburg, MD, USA) in which the upper and lower compartments were separated by 5 m pore polycarbonate polyvinylpyrrolidone-free filter (Neuro Probe, Gaithersburg, MD 20877, USA) to allow the cells to migrate actively through the pores. A suspension of 7.5 × 104 monocytes in 50 l was placed in the upper compartment, while various concentrations of NPS (over the range of 10−12 to 10−5 M) were placed in the lower compartment. A known stimulant of monocyte chemotaxis, fMLP (10−8 M) (Sigma, San Louis, MO, USA), was used as positive control. Monocyte migration was assessed after incubation with either chemoattractant for 90 min at 37 ◦ C in the presence of 5% CO2 . To evaluate the role of NPSR the NPSR antagonists [dCys(t Bu)5 ]NPS and [t Bu-d-Gly5 ]NPS (peptides) SHA 68 (nonpeptide) were added to the cells 30 min prior to NPS addition and during the incubation period. After incubation, the upper surfaces of the filters were scraped to remove nonmigrating cells. Filters were subsequently fixed and stained with Diff-Quik (Baxter, IL, USA). The number of migrating
17
Fig. 1. Chemotactic effects of NPS on human monocytes. fMLP, 10−8 M, has been used as a positive control. Data are expressed as mean ± S.E.M. of eight separate experiments. Statistical analysis was performed by one-way ANOVA followed by Bonferroni post hoc test. *p < 0.01 vs. control.
cells was determined in 5 fields per well, using light microscopy at 400× magnification. All experiments were performed in triplicate. NPS, [d-Cys(t Bu)5 ]NPS, [t Bu-d-Gly5 ]NPS and SHA 68, were synthesized in house as previously described [10,11,28]. 2.3. Statistical analysis The data are expressed as the number of migrating cells per field, (mean ± S.E.M. of n = 8 for agonist experiments and n = 4 for antagonist experiments). Data have been analyzed statistically by one-way or two-way ANOVA followed by Bonferroni post doc test for multiple comparison. Values of p < 0.05 were considered to be significant. 3. Results 3.1. Effect of NPS on human monocyte chemotaxis To evaluate the effects of NPS on monocyte chemotaxis a concentration-response curve was constructed over the range 10−12 M to 10−5 M. Control data were obtained in the presence of the medium alone (negative control) or 10−8 M fMLP (positive control). Results displayed in Fig. 1 show that in the presence of the medium alone (random migration) 20.6 ± 1.2 (mean ± S.E.M.) monocytes migrated. This increased to 98.9 ± 3.0 in the presence of 10−8 M fMLP. NPS induced a significant chemotactic effect starting from the concentration of 10−9 M (35.9 ± 4.7) with a peak effect at 10−5 M (80.0 ± 4.2) which is similar to the chemotactic response to 10−8 M fMLP. It is worthy of note that the concentration response curve to NPS is incomplete i.e. there is no plateau even at high peptide concentrations. 3.2. Effect of NPSR antagonists on fMLP- and NPS-induced chemotaxis This series of experiments was aimed at evaluating the involvement of NPSR in the chemotactic effect of NPS on human monocytes. Different NPSR antagonists of both peptide and nonpeptide nature were assessed alone or in the presence of NPS or fMLP. The peptide antagonists [d-Cys(t Bu)5 ]NPS and [t Bu-dGly5 ]NPS alone did not affect random migration at concentrations ranging from 10−3 to 10−5 M. The peptide antagonists did not modify the chemotactic action of 10−8 M fMLP. In contrast, both molecules, at the concentration of 10−3 M, significantly decreased the chemotactic effects of NPS at 10−5 and 10−7 M. No antagonistic effect was observed at the lower (10−5 M) concentration tested (Figs. 2 and 3). The nonpeptide antagonist SHA 68 alone, at 10−5 M, did not modify either random or the 10−8 fMLP-induced migration.
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Fig. 2. Effects of [d-Cys(t Bu)5 ]NPS on fMLP- or NPS-stimulated chemotaxis of human monocytes. Data, expressed as mean ± S.E.M. of four separate experiments, have been gathered in the figure for sake of clarity but statistically analyzed using separate two-way ANOVAs (one for each agonist used) followed by Bonferroni post hoc test. *p < 0.05 vs. control. # p < 0.05 vs. NPS.
Fig. 3. Effect of [t Bu-d-Gly5 ]NPS on fMLP or NPS-stimulated chemotaxis of human monocytes. Data, expressed as mean ± S.E.M. of four separate experiments, have been gathered in the figure for sake of clarity but statistically analyzed using separate two-way ANOVAs (one for each agonist used) followed by Bonferroni post hoc test. *p < 0.05 vs. control. # p < 0.05 vs. NPS.
At the same concentration, SHA 68 significantly decreased the chemotactic effects of NPS at 10−7 M (Fig. 4). 4. Discussion A major finding of the present work is the confirmation of the presence of functional NPS receptors in human monocytes. Moreover, NPS was demonstrated to behave as an effective chemoattractant agent for human monocytes. This effect is mediated by receptors of the NPSR type as demonstrated by classical pharmacological criteria, namely sensitivity to selective antagonists.
Fig. 4. Effect of SHA 68 on fLMP- or NPS-stimulated chemotaxis of human monocytes. Data, expressed as mean ± S.E.M. of four separate experiments, have been gathered in the figure for sake of clarity but statistically analyzed using separate two-way ANOVAs (one for each agonist used) followed by Bonferroni post hoc test. *p < 0.05 vs. control. # p < 0.05 vs. NPS.
As a standard proinflammatory agent we used the peptide fMLP which, through specific G-protein coupled receptors, activates multiple functional responses including chemotaxis [15]. In line with previous studies our data confirmed that fMLP acts as a powerful stimulant of monocyte chemotaxis [9]. We have shown here that NPS induces chemotaxis in human monocytes in a concentration dependent manner (range 10−9 M to 10−5 M). As described in the results section, the concentration–response curve to NPS is incomplete i.e. the Emax value could not be determined. The reasons for the unusual low potency of NPS measured under the present experimental conditions are not known. However it should be noted that statistically significant effects are elicited by the peptide in the nanomolar range and this is in line with most in vitro studies on NPS (see for instance [19,27]). To the best of our knowledge, the ability of NPS to stimulate human monocyte chemotaxis was not previously investigated while its action on mouse macrophage cell lines has already been documented [19]. Subsequently, we have used selective antagonists and have demonstrated that the chemotactic effect of NPS is mediated by selective activation of the NPSR protein. Our data show that SHA 68 displays a higher potency compared to [d-Cys(t Bu)5 ]NPS or [t Bu-dGly5 ]NPS. The higher potency of the nonpeptide antagonist is in line with results obtained in calcium mobilization studies performed at recombinant mouse [12] and rat receptors [24]. In contrast [t Bud-Gly5 ]NPS has been previously found to be more potent than [d-Cys(t Bu)5 ]NPS [24] while no difference was observed in our work. Moreover, the two peptidic antagonists are effective in counteracting NPS action only at the higher concentration used (i.e. 10−3 M). This might be explained by the low number of concentrations tested in the present study and/or by the limited difference in antagonist potency displayed by the two compounds (approximately threefold; see [24]). It is more difficult to explain the lack
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of inhibition observed at 10−5 M antagonist vs. 10−7 M agonist; a possible speculation may be related to the high density of NPS receptors on monocyte surface allowing a significant inhibition of migration only at high antagonist concentrations. It is also worth noting that SHA 68 showed antagonistic activity at concentrations (10−5 M) relatively higher in comparison to other in vitro experiments [25]. Interestingly, previous studies reported the presence of NPS and/or its receptor on the cells of the immune system [35]. For instance, in patients affected by inflammatory bowel disease, NPSR is present in inflammatory cells and its expression is increased upon monocyte and lymphocyte activation [7,27]. In human blood and sputum cells, monocytes/macrophages and eosinophils were identified as NPSR-positive [19]. It has previously been demonstrated using two different methods, that 1 M NPS increases migration in the mouse macrophage cell line RAW 264.7 [19]. Other studies have reported weak basal NPSR expression in epithelial cells of several organs and tissues (including small and large intestine), and an increase in expression during inflammation, such as inflammatory bowel disease and asthma [5]. However, it should be also mentioned that no differences were detected in the development of allergic lung disease in wild type and NPSR-deficient mice [1]. With respect to the pathophysiological role of NPS in inflammation, our data suggest that the inflammatory process can be sustained by the recruitment of monocytes/macrophages caused (at least in part) by an increase in NPS levels. Other studies hypothesized an involvement of the gain-of-function phenotype of Asn107Ile variant of the NPSR protein, which is associated with an increase in the intrinsic efficacy of NPS as demonstrated by in vitro studies in different cell lines [2]. Indeed, this NPSR isoform is associated with an increase in asthma susceptibility and other gastrointestinal inflammatory diseases [20]. For example, in the skin, neuropeptides act as mediators of the neuroimmune axis in an endocrine, paracrine and in some cases autocrine manner after being released also from sensory or autonomic nerve fibers during the inflammatory phase [18]. Neuropeptides like NPS can be secreted neuronally and modulate the immune response, as occurs in inflammatory diseases including asthma, arthritis, and complex regional pain syndromes [3]. Furthermore, high NPSR expression is found in murine brain and it has been suggested that NPSR might contribute to an inflammatory phenotype by neurally mediated mechanisms [17]. Thus, the NPS–NPSR system may play a role in regulating the pathways that link neuronal and immune responses. Our antagonist studies demonstrated that the receptor mediating monocyte chemotaxis induced by NPS is of the NPSR type. This evidence suggests that NPSR blockade in immune cells could be a useful strategy to reduce the inflammatory state which characterizes the above-mentioned pathologies. Acknowledgments We would like to thank Prof. Dave G. Lambert for his expertise in revising the manuscript as regards language and contents. References [1] Allen IC, Pace AJ, Jania LA, Ledford JG, Latour AM, Snouwaert JN, et al. Expression and function of NPSR1/GPRA in the lung before and after induction of asthmalike disease. Am J Physiol Lung Cell Mol Physiol 2006;291(5):L1005–17. [2] Bernier V, Stocco R, Bogusky MJ, Joyce JG, Parachoniak C, Grenier K, et al. Structure–function relationships in the neuropeptide S receptor: molecular consequences of the asthma-associated mutation N107I. J Biol Chem 2006;281(34):24704–12. [3] Brack A, Stein C. The role of the peripheral nervous system in immune cell recruitment. Exp Neurol 2003;184(1):44–9. [4] Camarda V, Rizzi A, Ruzza C, Zucchini S, Marzola G, Marzola E, et al. In vitro and in vivo pharmacological characterization of the neuropeptide S receptor antagonist [d-Cys(t Bu)5]neuropeptide S. J Pharmacol Exp Ther 2009;328(2):549–55.
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Contents lists available at SciVerse ScienceDirect
Peptides journal homepage: www.elsevier.com/locate/peptides
Neuropeptide S stimulates human monocyte chemotaxis via NPS receptor activation M. Filaferro a , C. Novi a , V. Ruggieri a , S. Genedani a , S. Alboni a , D. Malagoli b , G. Caló c , R. Guerrini d , G. Vitale a,∗ a
Department of Biomedical Sciences, Section of Pharmacology, University of Modena and Reggio Emilia, Via G. Campi 287, 41125 Modena, Italy Department of Biology, University of Modena and Reggio Emilia, Via G. Campi, 213/d, 41125 Modena, Italy c Department of Experimental and Clinical Medicine, Section of Pharmacology and Neuroscience Center and National Institute of Neuroscience, University of Ferrara, Via Fossato di Mortara 19, 44121 Ferrara, Italy d Department of Pharmaceutical Sciences and LTTA, University of Ferrara, Via Fossato di Mortara 19, 44100 Ferrara, Italy b
a r t i c l e
i n f o
Article history: Received 19 June 2012 Received in revised form 30 October 2012 Accepted 31 October 2012 Available online 8 November 2012 Keywords: Neuropeptide S fMLP Human NPS receptor Monocytes Chemotaxis NPSR antagonists
a b s t r a c t Neuropeptide S (NPS) produces several biological actions by activating a formerly orphan GPCR, now named NPS receptor (NPSR). It has been previously demonstrated that NPS stimulates murine leukocyte chemotaxis in vitro. In the present study we investigated the ability of NPS, in comparison with the proinflammatory peptide formyl-Met-Leu-Phe (fMLP), to stimulate human monocyte chemotaxis. At a concentration of 10−8 M fMLP significantly stimulated chemotaxis. NPS produced a concentration dependent chemotactic action over the concentration range 10−12 to 10−5 M. The NPSR antagonists [dCys(t Bu)5 ]NPS, [t Bu-d-Gly5 ]NPS and SHA 68 were used to pharmacologically characterize NPS action. Monocyte chemoattractant effect of NPS, but not fMLP, was completely blocked by either peptide antagonists or SHA with the nonpeptide molecule being more potent. None of the NPSR antagonists modified per se random cell migration. Thus, the present study demonstrated that NPS is able to stimulate human monocyte chemotaxis and that this effect is entirely due to selective NPSR activation. © 2012 Elsevier Inc. All rights reserved.
1. Introduction Neuropeptide S (NPS) is expressed in specific brain regions and has various effects on the central nervous system. There is strong evidence that NPS promotes anxiolytic-like effects in rodents [22,30,33]. In addition, NPS induces hyperlocomotion [33], increases wakefulness and suppresses all stages of sleep [33], suppresses food intake [6,8,26] and facilitates extinction of conditioned fear [13]. A bioinformatic analysis of current genome databases revealed that the NPS peptide precursor gene is present in all vertebrates, including rodents and pigs [32,34] with the exception of fish [21]. NPS activates its G protein-coupled receptor (NPSR) at low nanomolar concentrations and induces elevation of intracellular Ca2+ and adenosine 3 ,5 -cyclic monophosphate levels. Various findings suggest that the NPS–NPSR system may play a role in modulating innate immunity and chronic inflammatory diseases particularly of epithelial barriers. In fact, NPS and NPSR mRNAs are co-localized in the human bronchial and colon epithelia suggesting
Abbreviations: NPS, neuropeptide S; NPSR, NPS receptor; fMLP, formyl-Met-LeuPhe. ∗ Corresponding author. Tel.: +39 059 2055370; fax: +39 059 2055376. E-mail address: [email protected] (G. Vitale). 0196-9781/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.peptides.2012.10.013
that NPSR may be activated by autocrine or paracrine mechanisms [19,29]. Leukocyte migration into tissues represents a key process in the pathogenesis of inflammatory diseases. Data obtained in clinical trials have convincingly shown that inhibition of leukocyte migration into target organs represents an effective therapeutic approach for diseases in which inflammation has a noxious effect [23]. Interestingly, in vitro studies showed that NPS is able to modulate macrophage phagocytosis; indeed, prestimulation of mouse macrophage cell line RAW 264.7 with NPS resulted in a dose-dependent increase in phagocytosis of fluoresceinlabeled Escherichia coli. Furthermore, stimulation with NPS (1 M) increased cell migration and decreased cell adhesion, suggesting that NPS is a chemotactic agent to murine macrophages in vitro [19]. Studies related to pig immune system showed the presence of NPSR in immune tissues, including thymus, spleen, jejunal lymph nodes and soft palatine tonsils [34] and NPS was found to act as a modulator of lymphocyte proliferation. Moreover, evidence from human genetic studies demonstrated the association of NPSR gene polymorphisms with chronic inflammatory diseases of the respiratory [14] and gastrointestinal [7] system. NPS and its receptor are thought to have a role in asthma pathogenesis; single nucleotide polymorphisms within NPSR have been shown to be associated with an increased prevalence of asthma [2]. Peripheral tissues and
M. Filaferro et al. / Peptides 39 (2013) 16–20
epithelia of several organs express NPSR isoforms A and B, including the intestinal mucosa and the skin, and NPSR appears to be upregulated in inflammation [27]. Indeed, NPS increased the proliferation of splenic lymphocytes and enhanced pulmonary alveolar macrophage function. Although NPS alone did not show a relevant effect on the production of IL-1, IL-6 and TNF-␣, NPS could significantly enhance their production induced by LPS [35]. Thus, it seems that NPS was able to modulate the inflammatory response only in the presence of exogenous antigens such as LPS [35]. Chemotaxis is a fundamental process in which cells migrate directionally when they are exposed to external specific chemical gradients. It is exhibited by a wide variety of cells types and involves distinct strategies that depend on the cell and the environmental conditions. Several members of the neuropeptide family exert chemotactic actions on blood monocytes. Furthermore, the immune system influences the central nervous system. At a molecular level, neuro and immune signal molecules (hormones, neurotransmitters, neuropeptides, and cytokines) and their receptors are indeed members of the same superfamilies which enable the mutual neuroimmune communication [31]. In the present study, we investigated the ability of NPS to influence human monocyte chemotaxis, in comparison with the proinflammatory peptide formyl-Met-Leu-Phe (fMLP). Moreover, the NPSR peptide antagonists [d-Cys(t Bu)5 ]NPS [4], [t Bu-dGly5 ]NPS [24], and nonpeptide antagonist SHA 68 [16,25] were used in order to pharmacologically characterize the effect of NPS. 2. Materials and methods 2.1. Preparation of human monocytes Human mononuclear cells were separated by LympholyteH (Cederlane Burlington, NC, USA) gradient centrifugation of peripheral blood (collected into heparinated Vacutainers) obtained from healthy volunteers. After centrifugation (20 min at 800 × g), monocytes were removed from the interface between plasma and Lympholyte-H. Mononuclear cells were washed twice in PBS, resuspended in RPMI 1640 medium (EuroClone Milano, Italy) supplemented with 0.1% BSA (SERVA Electrophoresis, Heidelberg, Germany), and diluted to a final concentration of 1.5 × 106 monocytes/ml. 2.2. Monocyte migration assay Migration assays were performed by using 48-well microchemotaxis chambers (Boyden modified, Neuroprobe, Gaithersburg, MD, USA) in which the upper and lower compartments were separated by 5 m pore polycarbonate polyvinylpyrrolidone-free filter (Neuro Probe, Gaithersburg, MD 20877, USA) to allow the cells to migrate actively through the pores. A suspension of 7.5 × 104 monocytes in 50 l was placed in the upper compartment, while various concentrations of NPS (over the range of 10−12 to 10−5 M) were placed in the lower compartment. A known stimulant of monocyte chemotaxis, fMLP (10−8 M) (Sigma, San Louis, MO, USA), was used as positive control. Monocyte migration was assessed after incubation with either chemoattractant for 90 min at 37 ◦ C in the presence of 5% CO2 . To evaluate the role of NPSR the NPSR antagonists [dCys(t Bu)5 ]NPS and [t Bu-d-Gly5 ]NPS (peptides) SHA 68 (nonpeptide) were added to the cells 30 min prior to NPS addition and during the incubation period. After incubation, the upper surfaces of the filters were scraped to remove nonmigrating cells. Filters were subsequently fixed and stained with Diff-Quik (Baxter, IL, USA). The number of migrating
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Fig. 1. Chemotactic effects of NPS on human monocytes. fMLP, 10−8 M, has been used as a positive control. Data are expressed as mean ± S.E.M. of eight separate experiments. Statistical analysis was performed by one-way ANOVA followed by Bonferroni post hoc test. *p < 0.01 vs. control.
cells was determined in 5 fields per well, using light microscopy at 400× magnification. All experiments were performed in triplicate. NPS, [d-Cys(t Bu)5 ]NPS, [t Bu-d-Gly5 ]NPS and SHA 68, were synthesized in house as previously described [10,11,28]. 2.3. Statistical analysis The data are expressed as the number of migrating cells per field, (mean ± S.E.M. of n = 8 for agonist experiments and n = 4 for antagonist experiments). Data have been analyzed statistically by one-way or two-way ANOVA followed by Bonferroni post doc test for multiple comparison. Values of p < 0.05 were considered to be significant. 3. Results 3.1. Effect of NPS on human monocyte chemotaxis To evaluate the effects of NPS on monocyte chemotaxis a concentration-response curve was constructed over the range 10−12 M to 10−5 M. Control data were obtained in the presence of the medium alone (negative control) or 10−8 M fMLP (positive control). Results displayed in Fig. 1 show that in the presence of the medium alone (random migration) 20.6 ± 1.2 (mean ± S.E.M.) monocytes migrated. This increased to 98.9 ± 3.0 in the presence of 10−8 M fMLP. NPS induced a significant chemotactic effect starting from the concentration of 10−9 M (35.9 ± 4.7) with a peak effect at 10−5 M (80.0 ± 4.2) which is similar to the chemotactic response to 10−8 M fMLP. It is worthy of note that the concentration response curve to NPS is incomplete i.e. there is no plateau even at high peptide concentrations. 3.2. Effect of NPSR antagonists on fMLP- and NPS-induced chemotaxis This series of experiments was aimed at evaluating the involvement of NPSR in the chemotactic effect of NPS on human monocytes. Different NPSR antagonists of both peptide and nonpeptide nature were assessed alone or in the presence of NPS or fMLP. The peptide antagonists [d-Cys(t Bu)5 ]NPS and [t Bu-dGly5 ]NPS alone did not affect random migration at concentrations ranging from 10−3 to 10−5 M. The peptide antagonists did not modify the chemotactic action of 10−8 M fMLP. In contrast, both molecules, at the concentration of 10−3 M, significantly decreased the chemotactic effects of NPS at 10−5 and 10−7 M. No antagonistic effect was observed at the lower (10−5 M) concentration tested (Figs. 2 and 3). The nonpeptide antagonist SHA 68 alone, at 10−5 M, did not modify either random or the 10−8 fMLP-induced migration.
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Fig. 2. Effects of [d-Cys(t Bu)5 ]NPS on fMLP- or NPS-stimulated chemotaxis of human monocytes. Data, expressed as mean ± S.E.M. of four separate experiments, have been gathered in the figure for sake of clarity but statistically analyzed using separate two-way ANOVAs (one for each agonist used) followed by Bonferroni post hoc test. *p < 0.05 vs. control. # p < 0.05 vs. NPS.
Fig. 3. Effect of [t Bu-d-Gly5 ]NPS on fMLP or NPS-stimulated chemotaxis of human monocytes. Data, expressed as mean ± S.E.M. of four separate experiments, have been gathered in the figure for sake of clarity but statistically analyzed using separate two-way ANOVAs (one for each agonist used) followed by Bonferroni post hoc test. *p < 0.05 vs. control. # p < 0.05 vs. NPS.
At the same concentration, SHA 68 significantly decreased the chemotactic effects of NPS at 10−7 M (Fig. 4). 4. Discussion A major finding of the present work is the confirmation of the presence of functional NPS receptors in human monocytes. Moreover, NPS was demonstrated to behave as an effective chemoattractant agent for human monocytes. This effect is mediated by receptors of the NPSR type as demonstrated by classical pharmacological criteria, namely sensitivity to selective antagonists.
Fig. 4. Effect of SHA 68 on fLMP- or NPS-stimulated chemotaxis of human monocytes. Data, expressed as mean ± S.E.M. of four separate experiments, have been gathered in the figure for sake of clarity but statistically analyzed using separate two-way ANOVAs (one for each agonist used) followed by Bonferroni post hoc test. *p < 0.05 vs. control. # p < 0.05 vs. NPS.
As a standard proinflammatory agent we used the peptide fMLP which, through specific G-protein coupled receptors, activates multiple functional responses including chemotaxis [15]. In line with previous studies our data confirmed that fMLP acts as a powerful stimulant of monocyte chemotaxis [9]. We have shown here that NPS induces chemotaxis in human monocytes in a concentration dependent manner (range 10−9 M to 10−5 M). As described in the results section, the concentration–response curve to NPS is incomplete i.e. the Emax value could not be determined. The reasons for the unusual low potency of NPS measured under the present experimental conditions are not known. However it should be noted that statistically significant effects are elicited by the peptide in the nanomolar range and this is in line with most in vitro studies on NPS (see for instance [19,27]). To the best of our knowledge, the ability of NPS to stimulate human monocyte chemotaxis was not previously investigated while its action on mouse macrophage cell lines has already been documented [19]. Subsequently, we have used selective antagonists and have demonstrated that the chemotactic effect of NPS is mediated by selective activation of the NPSR protein. Our data show that SHA 68 displays a higher potency compared to [d-Cys(t Bu)5 ]NPS or [t Bu-dGly5 ]NPS. The higher potency of the nonpeptide antagonist is in line with results obtained in calcium mobilization studies performed at recombinant mouse [12] and rat receptors [24]. In contrast [t Bud-Gly5 ]NPS has been previously found to be more potent than [d-Cys(t Bu)5 ]NPS [24] while no difference was observed in our work. Moreover, the two peptidic antagonists are effective in counteracting NPS action only at the higher concentration used (i.e. 10−3 M). This might be explained by the low number of concentrations tested in the present study and/or by the limited difference in antagonist potency displayed by the two compounds (approximately threefold; see [24]). It is more difficult to explain the lack
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of inhibition observed at 10−5 M antagonist vs. 10−7 M agonist; a possible speculation may be related to the high density of NPS receptors on monocyte surface allowing a significant inhibition of migration only at high antagonist concentrations. It is also worth noting that SHA 68 showed antagonistic activity at concentrations (10−5 M) relatively higher in comparison to other in vitro experiments [25]. Interestingly, previous studies reported the presence of NPS and/or its receptor on the cells of the immune system [35]. For instance, in patients affected by inflammatory bowel disease, NPSR is present in inflammatory cells and its expression is increased upon monocyte and lymphocyte activation [7,27]. In human blood and sputum cells, monocytes/macrophages and eosinophils were identified as NPSR-positive [19]. It has previously been demonstrated using two different methods, that 1 M NPS increases migration in the mouse macrophage cell line RAW 264.7 [19]. Other studies have reported weak basal NPSR expression in epithelial cells of several organs and tissues (including small and large intestine), and an increase in expression during inflammation, such as inflammatory bowel disease and asthma [5]. However, it should be also mentioned that no differences were detected in the development of allergic lung disease in wild type and NPSR-deficient mice [1]. With respect to the pathophysiological role of NPS in inflammation, our data suggest that the inflammatory process can be sustained by the recruitment of monocytes/macrophages caused (at least in part) by an increase in NPS levels. Other studies hypothesized an involvement of the gain-of-function phenotype of Asn107Ile variant of the NPSR protein, which is associated with an increase in the intrinsic efficacy of NPS as demonstrated by in vitro studies in different cell lines [2]. Indeed, this NPSR isoform is associated with an increase in asthma susceptibility and other gastrointestinal inflammatory diseases [20]. For example, in the skin, neuropeptides act as mediators of the neuroimmune axis in an endocrine, paracrine and in some cases autocrine manner after being released also from sensory or autonomic nerve fibers during the inflammatory phase [18]. Neuropeptides like NPS can be secreted neuronally and modulate the immune response, as occurs in inflammatory diseases including asthma, arthritis, and complex regional pain syndromes [3]. Furthermore, high NPSR expression is found in murine brain and it has been suggested that NPSR might contribute to an inflammatory phenotype by neurally mediated mechanisms [17]. Thus, the NPS–NPSR system may play a role in regulating the pathways that link neuronal and immune responses. Our antagonist studies demonstrated that the receptor mediating monocyte chemotaxis induced by NPS is of the NPSR type. This evidence suggests that NPSR blockade in immune cells could be a useful strategy to reduce the inflammatory state which characterizes the above-mentioned pathologies. Acknowledgments We would like to thank Prof. Dave G. Lambert for his expertise in revising the manuscript as regards language and contents. References [1] Allen IC, Pace AJ, Jania LA, Ledford JG, Latour AM, Snouwaert JN, et al. Expression and function of NPSR1/GPRA in the lung before and after induction of asthmalike disease. Am J Physiol Lung Cell Mol Physiol 2006;291(5):L1005–17. [2] Bernier V, Stocco R, Bogusky MJ, Joyce JG, Parachoniak C, Grenier K, et al. Structure–function relationships in the neuropeptide S receptor: molecular consequences of the asthma-associated mutation N107I. J Biol Chem 2006;281(34):24704–12. [3] Brack A, Stein C. The role of the peripheral nervous system in immune cell recruitment. Exp Neurol 2003;184(1):44–9. [4] Camarda V, Rizzi A, Ruzza C, Zucchini S, Marzola G, Marzola E, et al. In vitro and in vivo pharmacological characterization of the neuropeptide S receptor antagonist [d-Cys(t Bu)5]neuropeptide S. J Pharmacol Exp Ther 2009;328(2):549–55.
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