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Neuropeptide Y receptor-specifically modulates human neutrophil function
Journal of Neuroimmunology 195 (2008) 88 – 95 www.elsevier.com/locate/jneuroim
Neuropeptide Y receptor-specifically modulates human neutrophil function Sammy Bedoui a,b,⁎, Andreas Kromer a,c , Thomas Gebhardt d , Roland Jacobs e , Kerstin Raber a,g , Mirja Dimitrijevic f , Jörn Heine c , Stephan von Hörsten a,g a Department of Functional and Applied Anatomy, Hannover Medical School, Hannover, Germany The Walter and Eliza Hall Institute of Medical Research, Immunology Division, Melbourne, Australia c Department of Anesthesiology, Hannover Medical School, Hannover, Germany d Department of Gastroenterology, Hannover Medical School, Hannover, Germany e Department of Clinical Immunology, Hannover Medical School, Hannover, Germany Immunology Research Center “Branislav Jankovic”, Institute of Immunology and Virology “Torlak”, Belgrade, Serbia g Experimental Therapy, Franz-Penzoldt-Center, Friedrich-Alexander-University, Erlangen, Germany b
f
Received 25 October 2007; received in revised form 30 January 2008; accepted 31 January 2008
Abstract Despite a continuously growing body of evidence highlighting the role of NPY in the immune system, surprisingly little is known about its ability to alter human leukocyte function. We therefore set out to examine NPY receptor expression and functional effects of NPY in freshly isolated human neutrophils. Our results not only demonstrate for the first time the presence of specific NPY receptors on human neutrophils, but also unveil of how these receptors differentially modulate critical functions of neutrophils such as phagocytosis of bacteria as well as the release of reactive oxygen species. © 2008 Elsevier B.V. All rights reserved. Keywords: Neuroimmune interactions; Neuropeptide Y (NPY); NPY receptors; Human neutrophils; Phagocytosis; Reactive oxygen species
1. Introduction Research in the last two decades has provided compelling evidence that the immune system not only functions as a selfregulatory and autonomous entity, but is subject to significant modulation by other supersystems, such as the nervous or the endocrine system (Bedoui et al., 2003a; Elenkov et al., 2000; Kohm and Sanders, 2001). Importantly, such interactions have been implicated in a growing number of diseases, e.g. autoimmunity (Bedoui et al., 2004), chronic inflammation (Marshall, 2004) and sepsis (Aldridge, 2002; Elenkov et al., 2000). A major pathway for the bidirectional interaction between the nervous and the immune system is provided by the sympathetic ⁎ Corresponding author. Immunology Division, The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3057, Australia. Tel.: +61 3 9345 2534. E-mail address: [email protected] (S. Bedoui). 0165-5728/$ - see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2008.01.012
nervous system (Elenkov et al., 2000; Kohm and Sanders, 2001). Though earlier studies focused mostly on the catecholamines, more recent work has established that other sympathetic transmitters, such as neuropeptide Y (NPY), also play a significant role within the neuroimmune crosstalk (Bedoui et al., 2003a). NPY, a 36 amino acid peptide, is released from sympathetic nerves innervating primary and secondary lymphoid organs (Lundberg et al., 1985) and modulates a variety of immunological functions, including chemotaxis (Straub et al., 2000), T lymphocyte differentiation (Kawamura et al., 1998; Levite and Chowers, 2001) and leukocyte migration (Bedoui et al., 2001). Notably, Hauser et al. (1993) demonstrated that NPY treatment prolongs the survival of endotoxemic rats, implying a protective role for NPY in sepsis. Though these initial studies attributed the protective-like action of NPY entirely to its complex cardiovascular function (Qureshi et al., 1998), we recently demonstrated that the reduced lethality is also associated with significantly decreased tissue infiltration of neutrophils and T lymphocytes due
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to increased adhesiveness to the marginal pool (Nave et al., 2004), suggesting that the immunomodulatory action of NPY is also relevant to sepsis. Neutrophils are an essential component of the body's first line of defense. With their ability to eliminate invading microbes e.g. by means of the secretion of highly reactive oxygen species and phagocytosis, neutrophils play a key role in the pathophysiology of sepsis (Aldridge, 2002). The production of oxygen species, often referred to as respiratory burst, is a tightly regulated mechanism, involving NADPH-oxidase and myeloperoxidase as key enzymes. Bacterial products such as fMLP and cytokines are potent activators of the respiratory burst. These factors initiate a cascade of intracellular events that particularly involve G-protein coupled receptors and protein kinase C activation (Quinn and Gauss, 2004). NPY also appears to be a modulator of the respiratory burst, as the fMLP-evoked respiratory burst is modulated by NPY in a dose-dependent manner both in vitro and in vivo (Hafström et al., 1993; von Hörsten et al., 1998). However, much of the interaction of NPY with neutrophils remains unknown. For example, it is unclear whether NPY also interferes with other critical neutrophil functions, such as phagocytosis, and what NPY receptors mediate such effects. Based on the demonstration of protective effects of NPY in experimental sepsis that also target neutrophils (Nave et al., 2004), and promising findings regarding the interplay between human neutrophils and NPY (Hafström et al., 1993), we set out to further characterize the effect of NPY on human neutrophils function. To this end, we examined freshly isolated human neutrophils for the expression of functional NPY receptors and assessed whether NPY modulates the phagocytotic abilities of the neutrophils. 2. Materials and methods 2.1. Samples and peptides For the in vitro experiments with human neutrophils, heparinized blood was collected from healthy volunteers. The institutional ethics committee approved the study and informed consent was obtained from the donors. NPY (Michel et al., 1998; Tatemoto et al., 1982; Tatemoto et al., 1985), pancreatic polypeptide (Michel et al., 1998), Leu31Pro34-NPY (Fuhlendorff et al., 1990), NPY13–36 (Doods et al., 1999; Michel et al., 1998; Schwartz et al., 1987) and D-Trp32 (Hwa et al., 1999) were obtained from PolyPeptide Laboratories (Wolfenbüttel, Germany). BIBO 3304 (Wieland et al., 1998) and BIIE 0246 (Doods et al., 1999) were kindly provided by Boehringer Ingelheim (Biberach, Germany). 2.2. Isolation of neutrophil granulocytes Venous blood samples (10 ml), anticoagulated with Lithiumheparin, were drawn from healthy blood donors in the morning. To obtain neutrophils, blood samples were underlayed with Ficoll-Paque (Amersham Biosciences, Uppsala, Sweden) and centrifuged for 30 min to separate neutrophils from peripheral blood mononuclear cells (PBMC). The granulocyte layer usually
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contained N98% granulocytes with b1% contamination with PBMC and we routinely retrieved 1–3 million granulocytes from an individual preparation that were then used for the assays described below. 2.3. PCR For RT-PCR neutrophils were separated from peripheral blood (40 ml) of healthy volunteers using a cell sorter gating on granulocytes according to the FSC versus SSC properties of the cells (FACStar plus, Becton Dickinson, Heidelberg, Germany). Since NPY receptors are abundantly expressed in the central nervous system, we used commercially available human brain suspensions as positive controls (BD Biosciences, Palo Alto, CA). RNA was extracted using the RNeasy spin column purification kit (Qiagen, Hilden, Germany), and concentrations were measured using a photometer (OD 260 nm). M-MLV reverse transcriptase (Invitrogen, Karlsruhe, Germany) was used to perform reverse transcription. Identical amounts of RNA were used. PCR reactions were performed in a 20 μl volume with 0.5 μM primers: Y1R sense 5′GTAGGTATTGCTGTGATTTGGG-3′ and antisense 5′CTCTGGAAGTTTTTGTTCA GGA-3′; Y2R sense 5′CCTACTGCTCCATCATCTTGC-3′ and antisense 5′GTAGTTGCTGTTCATCCAGCC-3′; Y4R sense 5′GTGTTTCAC AAGGGCACCTA-3′ and antisense 5′ TGCCACTTAGCCTCAGGGA-3′, Y5R (sense) 5′-AGCCATGTGCCATATCATGC-3′ and (anti-sense) 5′-GAGGCAGGATATACTGCACT-3′, NPY sense 5′AGCCATGTGCCATATTAGC3′ and antisense 5′GGCAGAATA TACTGAACTAGC-3′; β-tubulin rRNA sense 5′TTCCCTGG CCAGCTSAANGCNGAGCTNCGCAAG-3′ and antisense 5′-CATGCCCTCG CCNGTGTACCAGTGNANGAAGGC-3′, 1.5 mM MgCl2 and 10 μl Qiagen Mastermix. After 10 min denaturation at 95 °C, 32–38 cycles with 95 °C for 30 s, 54 °C (β-tubulin)/ 58 °C (Y1R, Y5R)/59 °C (Y4R)/61 °C (NPY, CD26) for 30 s, and 72 °C 60 s were performed. After PCR amplification, the reactions were analyzed by gel electrophoresis. Samples were diluted 1:2 with loading dye and loaded onto 2% agarose gels containing ethidium bromide. Gels were run for 45 min at 80 V. Expression of 18S served as positive control. Quantitative analysis of NPY receptor RNA was performed using a smart cycler (Cepheid, France) taking advantage of the QuantiTec SYBR Green™ technology (Qiagen). RNA from sorted neutrophils was isolated as described above. Each PCR cycle comprised melting at 95 °C for 15 s, annealing at a temperature specific for each gene (hCD26: 56 °C, hNPY: 54 °C, hNPYY1: 57.5 °C, hNPYY2: 58 °C, hNPYY4: 55 °C, hNPYY5: 56 °C, hGAPDH: 55 °C) for 30 s, and an extension at 72 °C for 30 s. Each PCR amplification was performed in triplicates. The optimal parameters for the PCR reactions were defined empirically. The purity of the amplified PCR products was verified by melting curves. hGAPDH was used as housekeeping gene. The specificity of the oligonucleotides (see above) used to amplify the NPY receptors was determined using cell lines transfected with specific cDNAs (SK-N-MC cells — Y1R; stably transfected HEC-1B — Y5; transiently transfected COS-7cells — Y2R and Y4R).
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2.4. Radio ligand binding For the radio ligand assay, granulocytes were enriched by Ficoll density gradient as described above. Five to seven million granulocytes were homogenized in 50 volumes of ice-cold Tris–HCl buffer (50 mM Tris, 100 mM NaCl, 5 mM KCl, pH 7.5) and centrifuged for 15 min at 48,000 g (0 °C). The resulting membranes were resuspended in 200 volumes of incubation buffer (50 mM Tris, 100 mM NaCl, 1 mM EGTA, 5 mM MgCl2 supplemented with 50 μM PMSF, 1% BSA, 0.025% Bacitracin and 1 μM phosphoramidon) and stored. The homogenate (0.1 ml) was incubated for 2 h at room temperature in the presence of 30 pM 125I-NPY and increasing concentrations of unlabeled NPY in a total volume of 0.25 ml. The incubation was stopped by centrifugation (10 min, 3000 g), the pellet washed with 0.25 ml of incubation buffer without resuspension, recentrifuged and membrane-bound radioactivity was counted. Non-specific binding was determined in the presence of 100 nM NPY. The binding assays were performed 3 to 6 times, each value in triplicate.
with 2 ml PBS and addition of PI (5 × 10− 5 mol/l) to stain the DNA of cells and bacteria, the samples were analyzed within 20 min by flow cytometry. A linear region on the sharp peak signal in the histogram of FL3 was set to discriminate leukocytes from bacteria. With this, linear region bacteria and cell debris were excluded by live gating during acquisition. In each sample 20,000 events were measured. FSC/SSC dot plots were used to gate neutrophils. The percentage of phagocytizing neutrophils was calculated after setting a quadrant region that was adjusted to the negative control. 2.7. Statistical analysis Data were analyzed using one-factor analyses of variance (ANOVA) for repeated measures and were followed by Fisher LSD post hoc analysis at specific dosages, if appropriate. Results are demonstrated as percentile change from controls. Values represent means ± SEM and asterisks in the figures indicate significant post hoc effects.
2.5. Assessment of respiratory burst Separated neutrophils were resuspended in PBS and incubated with 0.11 mM dihydrorhodamine (DHR, Sigma, Germany) at 37 °C. Various concentrations of NPY and NPYspecific agonists and antagonists were added. After 10 min of incubation the respiratory burst (RB) was initiated using fMLP (0.01 mmol l− 1, Sigma, Seelze, Germany). 10 μl of propidium iodine (PI, Sigma, Seelze, Germany) was included to differentiate between viable and dead cells. Fifteen minutes later, samples were put on ice to terminate the reaction. Cells were analyzed by flow cytometry (Coulter Epics, Krefeld, Germany) using the blue-green excitation light (488 nm argonion laser). Neutrophils were included by setting a polygonal gate in the forward (FSC) versus the sideward scatter signals (SSC). Twenty thousand events were included for each measurement. The DHR emission was filtered and measured with the green photomultiplier (FL1). The percentage of DHR positive cells in the test samples was then determined by counting the number of events above this marker position and dividing it with the whole events observed. PI emission was detected with the red photomultiplier (FL3). Results are expressed as percentile changes compared to unstimulated controls. 2.6. Phagocytosis assay Phagocytosis of opsonized Escherichia coli (Phagotest, Orpegen, Heidelberg, Germany) was assessed using a commercial test kit. Briefly, whole blood samples were incubated with 5 μl fluorescein isothiocyanate (FITC)-labeled opsonized E. coli (1 × 109/ml E. coli, Orpegen, Germany) at 37 °C for a period of 10 min. Negative controls were kept on ice. The cells were treated with a quenching solution to avoid false positive staining of bacteria adhered to the cell surface. After centrifuging and additional washing with PBS (250 g, 5 min, 4 °C), resuspension
Fig. 1. (A) Human neutrophils (PMN) fail to express NPY mRNA, but express mRNA encoding the Y1, Y2, Y4 and the Y5 receptors. For RT-PCR, human neutrophils were freshly isolated from peripheral blood of healthy volunteers using a cell sorter. A commercially available suspension of human brain was used as positive control. β-tubulin served as house keeping gene. One representative experiment out of three is shown. (B) Quantitative analysis of receptor expression before and after stimulation with 10 μM fMLP in comparison to PBMC and positive control levels (brain). PCR was performed with 38 cycles and the PCR products had the following sizes. Y1 receptor: 188 bp; Y2 receptor: 183 bp; Y4 receptor: 190 bp; Y5 receptor: 187. (C) Binding of 125I-labeled NPY to membranes from human neutrophils. IC50 was determined at 78 nM. Neutrophils were homogenized to obtain membrane suspensions. The membranes were incubated with 125I-labeled NPY (30 pM) and various concentrations of unlabeled NPY were added. Binding of 125I-labeled NPY is competed by native NPY, indicating the specificity of the binding. Each concentration was determined in triplicates.
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3. Results 3.1. Human neutrophils express NPY receptors A critical prerequisite for NPY to modulate human neutrophil function is the expression of functional NPY receptors. In a first step we investigated NPY receptor mRNA expression using RTPCR. Though separation of neutrophils by density gradients yields cell suspensions with sufficient purity for functional assays (b3% non-neutrophils), RT-PCR analysis was performed with sorted neutrophils to ensure maximal purity. As indicated in Fig. 1A, we found mRNA encoding the NPY receptor subtypes Y1, Y2, Y4 and Y5 in human neutrophils. Commercially available human brain suspensions were used as positive controls. To extent this analysis, we also performed real time PCR on enriched neutrophils before and after stimulation with fMLP and compared relative expression of the receptors with positive controls and peripheral blood mononuclear cells (PBMC). As shown in Fig. 1B, the expression hierarchy of the receptors followed the following order: Y4 receptor N Y1 and Y2 receptor N Y5 receptor. Whereas Y1, Y4 and Y5 receptor expression remained stable after stimulation with fMLP, Y2 receptor expression was significantly increased after stimulation. Even though the expression levels for the Y1, Y2 and Y4 receptor were comparable between neutrophils and PBMC, we found no relevant Y5 receptor expression in PBMC. In a second step we employed radio ligand binding studies to determine whether specific binding sites, indicative for the presence of NPY receptors, exist on the surface of the neutrophils. In these studies we detected binding of radiolabeled 125 I-NPY to the neutrophil membranes (Fig. 1C). Binding of 125I-NPY to the membranes can be competed by adding increasing concentrations of unlabeled NPY, demonstrating that binding of NPY to the human neutrophils is specific. Thus, based on RT-PCR and radio ligand binding studies, we show here that human neutrophils possess NPY receptors. 3.2. Priming of the fMLP-induced respiratory burst is mediated via the NPY Y5 receptor subtype Given that human neutrophils express NPY receptors we next explored whether these receptors are functional. To this end, we stimulated freshly isolated human neutrophils with E. coli and fMLP in vitro, and determined their production of reactive oxygen species by flow cytometry. NPY significantly increased the respiratory burst induced by fMLP in a dose-dependent manner (Fig. 2A), demonstrating that the NPY receptors on the neutrophils are fully functional. The results from the RT-PCR suggested a role for the Y1, Y2, Y4 and Y5 receptors in the priming of the respiratory burst elicited by NPY. To clarify the functional involvement of these receptor subtypes, we employed an array of different NPY receptor-specific agonists and antagonists. Among the tested agonists, only Leu31Pro34-NPY and D-Trp32 induced a significant intensification of the respiratory burst that was comparable to the action of native NPY (Fig. 2B). With Leu31Pro34-NPY being a selective agonist at the Y1 and the Y5 receptor, and D-Trp32 selectively activating the Y5 receptor,
Fig. 2. A. NPY intensifies the fMLP-induced respiratory burst in a dose-dependent fashion. Neutrophils were isolated from peripheral blood of healthy volunteers and were stimulated with 10 μM fMLP in the presence of various concentrations of NPY. B and C. Pharmacological assessment of the involvement of different NPY receptor subtypes in the fMLP-induced respiratory burst. Neutrophils were isolated from peripheral blood of healthy volunteers and were stimulated in vitro with 10 μM fMLP in the presence of different NPY receptor-specific agonists (B). Only NPY (10− 5 M), Leu31-Pro34-NPY (10− 5 M) and D-Trp32 (10− 5 M) significantly intensified the respiratory burst, suggesting that the effect is mediated by the Y1 and/or the Y5 receptor. Combining NPY with the Y1 receptor antagonist BIBO 3304 (10− 4 M) failed to alter the intensification elicited by NPY (C), demonstrating that Y1 receptor activation plays no significant role in the intensification of the respiratory burst by NPY. The respiratory burst was assessed using a flow cytometer. Values are expressed as relative changes from controls. Results are the mean ± SEM of n = 5 experiments.
these data suggest that the priming of the respiratory burst is mediated via the Y5 receptor subtype. Formally, the intensification of the respiratory burst by Leu31Pro34-NPY could also indicate additional Y1 receptor mediated effects. This possibility, however, is ruled out by the observation that a combination of the selective Y1 receptor antagonist BIBO3304 and native NPY results in a comparable intensification of the respiratory burst as elicited by both native NPY (Fig. 2C) and D-Trp32. Thus, the NPY-induced intensification of the respiratory burst by human neutrophils is mediated via the Y5 receptor subtype. 3.3. Both the Y1 and the Y2 receptor subtype modulate phagocytosis by neutrophils Another hallmark of neutrophil function is their ability to ingest microorganisms and particles. To evaluate whether NPY
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also interferes with this important neutrophil function, we analyzed phagocytosis of fluorescent-labeled E. coli by flow cytometry. Fig. 3A demonstrates a dose-dependent, bimodal
effect of NPY on neutrophil phagocytosis: whereas low concentrations significantly potentiate phagocytosis, increasing dosages eventually inhibit phagocytosis by about 25%. It was interesting to assess whether this effect also depends on Y5 receptor activation or whether other receptor subtypes are involved. Notably, phagocytosis can only be inhibited using Leu31Pro34-NPY and the Y2 receptor-selective agonist NPY13–36 (Fig. 3B), suggesting a combined involvement of the Y1 and the Y2 receptor. Even though the effects of Leu31Pro34-NPY could formally also be attributed to Y5 activation, this is highly unlikely, as the Y5 receptor agonist D-Trp32 had no effect. Again, we reevaluated these effects by combining native NPY with selective antagonists of the Y1 and the Y2 receptor. In the presence of both the Y1 receptor antagonist BIBO3304 and the Y2 receptor antagonist BIIE3246 (Fig. 3C), NPY fails to significantly inhibit phagocytosis. Thus, these findings demonstrate that NPY modulates phagocytosis via combined activation of the Y1 and the Y2 receptor. 4. Discussion
Fig. 3. A. Phagocytosis of fluorescence-labeled E. coli by human neutrophils is modulated by NPY. Neutrophils, isolated from peripheral blood of healthy volunteers, were incubated with FITC-labeled E. coli for 10 min at 37 °C. Cells were washed and binding of labeled E. coli to the cell membrane was avoided using a quenching solution. FITC-labeled E. coli were detected by flow cytometry. B and C. Pharmacological assessment of the involvement of different NPY receptor subtypes in the modulation of phagocytosis. Only NPY (10− 5 M), Leu31-Pro34NPY (10− 5 M) and NPY13–36 (10− 5 M) significantly inhibited phagocytosis, suggesting a combined action via the Y1 and the Y2 receptor (B). As the inhibition could be abrogated by addition of the Y1 receptor antagonist BIBO 3304 (10− 4M) and the Y2 receptor antagonist BIIE3226 (C), it is further substantiated that the ability of NPY to modulate phagocytosis is mediated via a combined interplay at the Y1 and the Y2 receptor subtype. Values are expressed as relative changes from controls. Results are the mean ± SEM of n = 5 experiments.
In the present study we have demonstrated that human neutrophils possess functional NPY receptors. Importantly, neutrophil function is regulated by NPY in a complex manner with different NPY receptor subtypes responsible for particular functions. Activation of the Y5 receptor potentiates the production of reactive oxygen species elicited by fMLP and parallel Y1 and Y2 receptor stimulation modifies phagocytosis of E. coli. A critical prerequisite for leukocytes to respond to signals from the nervous system is the expression of specific receptors for neurotransmitters or neuropeptides on their surfaces. Therefore, a major aim of the present study was to determine whether NPY receptors are present on human neutrophils. Our qualitative and quantitative PCR results revealed the presence of mRNA encoding for the Y1, Y2, Y4 and Y5 receptors in human neutrophils and established differential expression patterns for the respective receptors. Notably, when compared to a commercially available human brain suspension, expression levels of all NPY receptors were higher in neutrophils. As mRNA expression does not necessarily imply that a functional receptor protein is also present on the cell surface, we additionally employed radio ligand studies to investigate whether specific binding sites for NPY are present on neutrophils. 125 I-labeled NPY bound to membrane suspensions prepared from neutrophils and these binding sites were shown to be specific, as increasing amounts of unlabeled NPY displaced radio-labeled NPY in a dose-dependent manner. The competition curve followed a classical sigmoid curve and the IC50 was determined as 78 nM. Given this 100-fold higher IC50 value of the neutrophils compared to transfected cell lines (Fabry et al., 2000), it appears that the NPY receptors are not very densely distributed on neutrophils. Low-level expression also explains as to why other less sensitive methods of tracking NPY receptors expression with radio ligands (3H) produced inconclusive results (data not shown). Nevertheless, based on the presence of NPY receptor mRNA in neutrophils and the
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results from the binding study, we conclude that human neutrophils express NPY receptors. To assess the functional capacity of the expressed NPY receptors we employed two different in vitro assays. The production of reactive oxygen species and the phagocytosis of invading microbes are essential for the neutrophil to defend the body against invading microbes (Aldridge, 2002; Quinn and Gauss, 2004). Therefore, we tested the effect of NPY on the respiratory burst induced by fMLP stimulation in vitro and found that NPY potentiates this critical neutrophil function. Notably, not only the respiratory burst is significantly altered by NPY, also phagocytosis of fluorescence-labeled E. coli is subject to significant modulation by NPY. As both of the effects elicited by NPY follow a clear dose-dependency, the results prove that the detected NPY receptors on human neutrophils are functionally active. Despite the demonstration of NPY receptor mRNA, NPYspecific binding sites and functional in vitro effects of NPY on human neutrophils, the findings discussed so far fail to elucidate whether all or only some of the NPY receptor subtypes detected in PCR are actually involved in these effects. Therefore, we employed an array of different NPY-specific agonists and antagonists to determine pharmacologically which of the NPY receptor subtypes mediate the functional effects of NPY on the respiratory burst and phagocytosis. The intensification of the respiratory burst by NPY can be mimicked by the Y1 and Y5 receptor-specific agonist Leu31-Pro34-NPY and the Y5 receptorselective peptide D-Trp32, suggesting that the Y5 receptor mediates this intensification. Though Leu31-Pro34-NPY also exerts agonistic activities at the Y1 receptor, the possibility of an additional involvement of the Y1 receptor is excluded by our finding that NPY still potentiates the respiratory burst when the Y1 receptor is pharmacologically blocked by BIBO 3304. The pharmacological data, therefore, demonstrates that NPY potentiates the respiratory burst via activation of the Y5 receptor. What makes this finding particularly interesting is the observation that the expression levels for the Y5 receptor are rather low when compared to the other receptors, suggesting that this receptor is very efficient at modulating one of the most critical functions of neutrophils. Using a similar pharmacological approach, we also analyzed which receptor subtypes mediated the effects of NPY on phagocytosis. Interestingly, the results revealed a different pattern as both Leu31-Pro34-NPY and the Y2 receptor-selective agonist NPY13–36 mimic the inhibition of phagocytosis. Again we also used the combination of NPY and Y1 and Y2 receptor antagonists to further evaluate the role of these receptors in phagocytosis. Under both conditions the inhibition of phagocytosis by NPY is abrogated, indicating that both the Y1 and the Y2 receptor subtype mediated this effect. This finding in combination with the absence of any effects of D-Trp32 also argues against a role for the Y5 receptor that could formally be implicated by the action of Leu31-Pro34-NPY. Thus, the action of NPY on phagocytosis involves activation of the Y1 and the Y2 receptor. A more precise look at the data reveals some interesting additional details. Even though the Y1/Y5 receptor agonist Leu31-Pro34-NPY inhibits phagocytosis as potently as
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NPY, the combination of NPY with either a Y1 or a Y2 receptor antagonist — which should result in the stimulation of the Y1 and the Y2 receptor respectively — failed to significantly inhibit phagocytosis. To explain this apparent contradiction, it has to be taken into consideration that even though Leu31-Pro34NPY and NPY13–36 act as potent selective agonists at their respective receptors (Doods et al., 1999; Fuhlendorff et al., 1990), they still have the ability to activate other NPY receptors with a much lower affinity (Michel et al., 1998). Thus, in the situation where either Leu31-Pro34-NPY or NPY13–36 was added alone, they also exhibited a partial — though weak — agonism at the Y2 or Y1 receptor respectively. Consistent with this notion, addition of NPY in the presence of a Y1 or Y2 antagonist, where a partial agonism at the other receptor is ruled out, proved inefficient at inhibiting phagocytosis. This suggests that both the Y1 and Y2 receptor have to be activated in parallel for NPY to exert its full inhibitory activity on phagocytosis. In the light of a potent upregulation of the Y2 receptor upon stimulation (Fig. 1B) and the fact that the blockade of the Y1 receptor was not quite as efficient in restoring phagocytosis (Fig. 3C), it appears that the Y2 receptor delivers the stronger signal during this interplay. It will be exciting for future studies to unravel the molecular mechanism underlying this complex interaction. Even though the Y4 receptor was detected on the mRNA level and in comparison to the other receptor, was expressed at the highest level, the absence of any functional effect of PP, which preferentially activates the Y4 receptor, argues against any functional effects of the Y4 receptor protein on phagocytosis and the respiratory burst of human neutrophils. Verifying these findings by detecting the very receptor proteins on the surface of the neutrophils by flow cytometry, for example, is at present not possible due to the absence of monoclonal antibodies for NPY receptors. We are currently undertaking attempts to generate such antibodies. Taken together, the PCR results, the radio ligand binding studies and the in vitro effects demonstrate that human neutrophils express fully functional Y1, Y2 and Y5 receptor subtypes. Formally, this study cannot prove that the observed receptor-specific effects of NPY on differential neutrophil function are entirely the consequence of a direct interaction between NPY and neutrophils, as the methodological approach of using fresh human neutrophils always has the limitation that some contaminating blood cells are present in the preparations. Given that receptor expression levels on the neutrophils were either similar (for the Y1, Y2 and Y4 receptor) or higher (for the Y5 receptor) when compared to PBMC, indicates that the NPY receptors are expressed on a higher level in neutrophils than in the heterogeneous PBMC population, pointing towards direct rather than indirect effects. Further studies are necessary to absolutely exclude indirect effects. These studies extend our previous findings demonstrating the presence of functional Y1 receptors on rat PBMC (Bedoui et al., 2002a,b) and mouse T lymphocytes (Bedoui et al., 2003b; Petitto et al., 1994). With previous reports showing that human lymphocytes and monocytes produce NPY (Schwarz et al., 1994) on the one hand and the presence of functional NPY receptors on human neutrophils on the other, it was possible that human neutrophils
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also produce NPY, which in turn might be involved in an autocrine feedback loop. However, we found no support in favor of this hypothesis, as neither NPY mRNA was detectable in resting human neutrophils as well after 24 h of in vitro stimulation with fMLP, nor did the Y1 and Y2 receptor antagonists exert any effect on the neutrophils in vitro (data not shown). So, we are left with a complex picture that very much depends on which receptor is activated and at what concentration NPY is present. In low doses, NPY increases the phagocytosis of E. coli without changing the respiratory burst. However, in higher concentration NPY not only inhibits phagocytosis, but also augments the respiratory burst. Thus, it appears that NPY has the ability to switch the way a neutrophil deals with invading pathogens. When NPY is present at low doses, the pathogen is taken up and intracellular mechanisms are in charge of its elimination. Conversely, in microenvironments of higher NPY concentrations, the neutrophil allows fewer pathogens to be ingested and rather upregulates its ability to destroy the pathogen by extracellular means. The latter way of action might be particularly important in situations where large numbers of pathogens are encountered, ensuring that other cells, such as professional antigen presenting cells, can access the pathogen for antigen display to branches of the adaptive immune system. These findings also have interesting clinical implications. Septicaemia, during which the uncontrolled dissemination of pathogens and their products severely compromises the function of the immune system, still poses a major clinical problem and is associated with high mortality (Sessler et al., 2004). Over the last years, it has been conclusively shown that, in fact, the sympathetic nervous system also plays an important role during septicaemia through its effects on the immune system. Whereas this action has been traditionally attributed to the function of catecholamines (Kohm and Sanders, 2001), several lines of evidence also indicate that NPY can exert such protective-like effects during experimental septicaemia (Hauser et al., 1993; Nave et al., 2004). Given that patients with septicaemia often develop a functional resistance to exogenous catecholamines, the findings outlined in this study, might provide a first step towards the clinical utilization of NPY in patients with septicaemia in the future. Acknowledgements The excellent technical assistance of S. Kuhlmann and G. Peper, and the correction of the English by S. Fryk are very much appreciated. The authors would also like to thank Dr. R. Pabst for critical review of the manuscript. References Aldridge, A.J., 2002. Role of the neutrophil in septic shock and the adult respiratory distress syndrome. Eur. J. Surg. 168, 204–214. Bedoui, S., Kuhlmann, S., Nave, H., Drube, J., Pabst, R., von Hörsten, S., 2001. Differential effects of neuropeptide Y (NPY) on leukocyte subsets in the blood: mobilization of B-1-like B-lymphocytes and activated monocytes. J. Neuroimmunol. 117, 125–132. Bedoui, S., Lechner, S., Gebhardt, T., Nave, H., Beck-Sickinger, A.G., Straub, R.H., Pabst, R., von Hörsten, S., 2002a. NPY modulates epinephrine-induced
leukocytosis via Y-1 and Y-5 receptor activation in vivo: sympathetic cotransmission during leukocyte mobilization. J. Neuroimmunol. 132, 25–33. Bedoui, S., Lechner, S., Gebhardt, T., Nave, H., Beck-Sickinger, A.G., Straub, R.H., Pabst, R., von Hörsten, S., 2002b. NPY modulates epinephrine-induced leukocytosis via Y-1 and Y-5 receptor activation in vivo: sympathetic cotransmission during leukocyte mobilization. J. Neuroimmunol. 132, 25–33. Bedoui, S., Kawamura, N., Straub, R.H., Pabst, R., Yamamura, T., von Hörsten, S., 2003a. Relevance of neuropeptide Y for the neuroimmune crosstalk. J. Neuroimmunol. 134, 1–11. Bedoui, S., Miyake, S., Lin, Y., Miyamoto, K., Oki, S., Kawamura, N., BeckSickinger, A., von Hörsten, S., Yamamura, T., 2003b. Neuropeptide Y (NPY) suppresses experimental autoimmune encephalomyelitis: NPY1 receptorspecific inhibition of autoreactive Th1 responses in vivo. J. Immunol. 171, 3451–3458. Bedoui, S., Miyake, S., Straub, R.H., von Hörsten, S., Yamamura, T., 2004. More sympathy for autoimmunity with neuropeptide Y? Trends Immunol. 25, 508–512. Doods, H., Gaida, W., Wieland, H.A., Dollinger, H., Schnorrenberg, G., Esser, F., Engel, W., Eberlein, W., Rudolf, K., 1999. BIIE0246: a selective and high affinity neuropeptide Y Y(2) receptor antagonist. Eur. J. Pharmacol. 384, R3–R5. Elenkov, I.J., Wilder, R.L., Chrousos, G.P., Vizi, E.S., 2000. The sympathetic nerve—an integrative interface between two supersystems: the brain and the immune system. Pharmacol. Rev. 52, 595–638. Fabry, M., Cabrele, C., Hocker, H., Beck-Sickinger, A.G., 2000. Differently labeled peptide ligands for rapid investigation of receptor expression on a new human glioblastoma cell line. Peptides 21, 1885–1893. Fuhlendorff, J., Gether, U., Aakerlund, L., Langeland-Johansen, N., Thogersen, H., Melberg, S.G., Olsen, U.B., Thastrup, O., Schwartz, T.W., 1990. [Leu31, Pro34]neuropeptide Y: a specific Y1 receptor agonist. Proc. Natl. Acad. Sci. U. S. A. 87, 182–186. Hafström, I., Ringertz, B., Lundeberg, T., Palmblad, J., 1993. The effect of endothelin, neuropeptide Y, calcitonin gene-related peptide and substance P on neutrophil functions. Acta Physiol. Scand. 148, 341–346. Hauser, G.J., Myers, A.K., Dayao, E.K., Zukowska-Grojec, Z., 1993. Neuropeptide Y infusion improves hemodynamics and survival in rat endotoxic shock. Am. J. Physiol. 265, H1416–H1423. Hwa, J.J., Witten, M.B., Williams, P., Ghibaudi, L., Gao, J., Salisbury, B.G., Mullins, D., Hamud, F., Strader, C.D., Parker, E.M., 1999. Activation of the NPY Y5 receptor regulates both feeding and energy expenditure. Am. J. Physiol. 277, R1428–R1434. Kawamura, N., Tamura, H., Obana, S., Wenner, M., Ishikawa, T., Nakata, A., Yamamoto, H., 1998. Differential effects of neuropeptides on cytokine production by mouse helper T cell subsets. Neuroimmunomodulation 5, 9–15. Kohm, A.P., Sanders, V.M., 2001. Norepinephrine and beta 2-adrenergic receptor stimulation regulate CD4+ T and B lymphocyte function in vitro and in vivo. Pharmacol. Rev. 53, 487–525. Levite, M., Chowers, Y., 2001. Nerve-driven immunity: neuropeptides regulate cytokine secretion of T cells and intestinal epithelial cells in a direct, powerful and contextual manner. Ann. Oncol. 12 (Suppl 2), S19–S25. Lundberg, J.M., Martinsson, A., Hemsen, A., Theodorsson-Norheim, E., Svedenhag, J., Ekblom, B., Hjemdahl, P., 1985. Co-release of neuropeptide Y and catecholamines during physical exercise in man. Biochem. Biophys. Res. Commun. 133, 30–36. Marshall, G.D., 2004. Neuroendocrine mechanisms of immune dysregulation: applications to allergy and asthma. Ann. Allergy Asthma Immun. 93, S11–S17. Michel, M.C., Beck-Sickinger, A., Cox, H., Doods, H.N., Herzog, H., Larhammar, D., Quirion, R., Schwartz, T., Westfall, T., 1998. XVI. International Union of Pharmacology recommendations for the nomenclature of neuropeptide Y, peptide YY, and pancreatic polypeptide receptors. Pharmacol. Rev. 50, 143–150. Nave, H., Bedoui, S., Moenter, F., Steffens, J., Felies, M., Gebhardt, T., Straub, R.H., Pabst, R., Dimitrijevic, M., Stanojevic, S., von Hörsten, S., 2004. Reduced tissue immigration of monocytes by neuropeptide Y during endotoxemia is associated with Y2 receptor activation. J. Neuroimmunol. 155, 1–12. Petitto, J.M., Huang, Z., McCarthy, D.B., 1994. Molecular cloning of NPY-Y1 receptor cDNA from rat splenic lymphocytes: evidence of low levels of
S. Bedoui et al. / Journal of Neuroimmunology 195 (2008) 88–95 mRNA expression and [125I]NPY binding sites. J. Neuroimmunol. 54, 81–86. Quinn, M.T., Gauss, K.A., 2004. Structure and regulation of the neutrophil respiratory burst oxidase: comparison with nonphagocyte oxidases. J. Leukoc. Biol. 76, 760–781. Qureshi, N.U., Dayao, E.K., Shirali, S., Zukowska-Grojec, Z., Hauser, G.J., 1998. Endogenous neuropeptide Y mediates vasoconstriction during endotoxic and hemorrhagic shock. Regul. Pept. 75-76, 215–220. Schwartz, T.W., Sheikh, S.P., O'Hare, M.M., 1987. Receptors on phaeochromocytoma cells for two members of the PP-fold family—NPY and PP. FEBS Lett. 225, 209–214. Schwarz, H., Villiger, P.M., von Kempis, J., Lotz, M., 1994. Neuropeptide Y is an inducible gene in the human immune system. J. Neuroimmunol. 51, 53–61. Sessler, C.N., Perry, J.C., Varney, K.L., 2004. Management of severe sepsis and septic shock. Curr. Opin. Crit. Care 10, 354–363.
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Straub, R.H., Mayer, M., Kreutz, M., Leeb, S., Schölmerich, J., Falk, W., 2000. Neurotransmitters of the sympathetic nerve terminal are powerful chemoattractants for monocytes. J. Leukoc. Biol. 67, 553–558. Tatemoto, K., Carlquist, M., Mutt, V., 1982. Neuropeptide Y—a novel brain peptide with structural similarities to peptide YY and pancreatic polypeptide. Nature 296, 659–660. Tatemoto, K., Siimesmaa, S., Jornvall, H., Allen, J.M., Polak, J.M., Bloom, S.R., Mutt, V., 1985. Isolation and characterization of neuropeptide Y from porcine intestine. FEBS Lett. 179, 181–184. von Hörsten, S., Ballof, J., Helfritz, F., Nave, H., Meyer, D., Schmidt, R.E., Stalp, M., Klemm, A., Tschernig, T., Pabst, R., 1998. Modulation of innate immune functions by intracerebroventricularly applied neuropeptide Y: dose and time dependent effects. Life Sci. 63, 909–922. Wieland, H.A., Engel, W., Eberlein, W., Rudolf, K., Doods, H.N., 1998. Subtype selectivity of the novel nonpeptide neuropeptide Y Y1 receptor antagonist BIBO 3304 and its effect on feeding in rodents. Br. J. Pharmacol. 125, 549–555.
Neuropeptide Y receptor-specifically modulates human neutrophil function Sammy Bedoui a,b,⁎, Andreas Kromer a,c , Thomas Gebhardt d , Roland Jacobs e , Kerstin Raber a,g , Mirja Dimitrijevic f , Jörn Heine c , Stephan von Hörsten a,g a Department of Functional and Applied Anatomy, Hannover Medical School, Hannover, Germany The Walter and Eliza Hall Institute of Medical Research, Immunology Division, Melbourne, Australia c Department of Anesthesiology, Hannover Medical School, Hannover, Germany d Department of Gastroenterology, Hannover Medical School, Hannover, Germany e Department of Clinical Immunology, Hannover Medical School, Hannover, Germany Immunology Research Center “Branislav Jankovic”, Institute of Immunology and Virology “Torlak”, Belgrade, Serbia g Experimental Therapy, Franz-Penzoldt-Center, Friedrich-Alexander-University, Erlangen, Germany b
f
Received 25 October 2007; received in revised form 30 January 2008; accepted 31 January 2008
Abstract Despite a continuously growing body of evidence highlighting the role of NPY in the immune system, surprisingly little is known about its ability to alter human leukocyte function. We therefore set out to examine NPY receptor expression and functional effects of NPY in freshly isolated human neutrophils. Our results not only demonstrate for the first time the presence of specific NPY receptors on human neutrophils, but also unveil of how these receptors differentially modulate critical functions of neutrophils such as phagocytosis of bacteria as well as the release of reactive oxygen species. © 2008 Elsevier B.V. All rights reserved. Keywords: Neuroimmune interactions; Neuropeptide Y (NPY); NPY receptors; Human neutrophils; Phagocytosis; Reactive oxygen species
1. Introduction Research in the last two decades has provided compelling evidence that the immune system not only functions as a selfregulatory and autonomous entity, but is subject to significant modulation by other supersystems, such as the nervous or the endocrine system (Bedoui et al., 2003a; Elenkov et al., 2000; Kohm and Sanders, 2001). Importantly, such interactions have been implicated in a growing number of diseases, e.g. autoimmunity (Bedoui et al., 2004), chronic inflammation (Marshall, 2004) and sepsis (Aldridge, 2002; Elenkov et al., 2000). A major pathway for the bidirectional interaction between the nervous and the immune system is provided by the sympathetic ⁎ Corresponding author. Immunology Division, The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3057, Australia. Tel.: +61 3 9345 2534. E-mail address: [email protected] (S. Bedoui). 0165-5728/$ - see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2008.01.012
nervous system (Elenkov et al., 2000; Kohm and Sanders, 2001). Though earlier studies focused mostly on the catecholamines, more recent work has established that other sympathetic transmitters, such as neuropeptide Y (NPY), also play a significant role within the neuroimmune crosstalk (Bedoui et al., 2003a). NPY, a 36 amino acid peptide, is released from sympathetic nerves innervating primary and secondary lymphoid organs (Lundberg et al., 1985) and modulates a variety of immunological functions, including chemotaxis (Straub et al., 2000), T lymphocyte differentiation (Kawamura et al., 1998; Levite and Chowers, 2001) and leukocyte migration (Bedoui et al., 2001). Notably, Hauser et al. (1993) demonstrated that NPY treatment prolongs the survival of endotoxemic rats, implying a protective role for NPY in sepsis. Though these initial studies attributed the protective-like action of NPY entirely to its complex cardiovascular function (Qureshi et al., 1998), we recently demonstrated that the reduced lethality is also associated with significantly decreased tissue infiltration of neutrophils and T lymphocytes due
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to increased adhesiveness to the marginal pool (Nave et al., 2004), suggesting that the immunomodulatory action of NPY is also relevant to sepsis. Neutrophils are an essential component of the body's first line of defense. With their ability to eliminate invading microbes e.g. by means of the secretion of highly reactive oxygen species and phagocytosis, neutrophils play a key role in the pathophysiology of sepsis (Aldridge, 2002). The production of oxygen species, often referred to as respiratory burst, is a tightly regulated mechanism, involving NADPH-oxidase and myeloperoxidase as key enzymes. Bacterial products such as fMLP and cytokines are potent activators of the respiratory burst. These factors initiate a cascade of intracellular events that particularly involve G-protein coupled receptors and protein kinase C activation (Quinn and Gauss, 2004). NPY also appears to be a modulator of the respiratory burst, as the fMLP-evoked respiratory burst is modulated by NPY in a dose-dependent manner both in vitro and in vivo (Hafström et al., 1993; von Hörsten et al., 1998). However, much of the interaction of NPY with neutrophils remains unknown. For example, it is unclear whether NPY also interferes with other critical neutrophil functions, such as phagocytosis, and what NPY receptors mediate such effects. Based on the demonstration of protective effects of NPY in experimental sepsis that also target neutrophils (Nave et al., 2004), and promising findings regarding the interplay between human neutrophils and NPY (Hafström et al., 1993), we set out to further characterize the effect of NPY on human neutrophils function. To this end, we examined freshly isolated human neutrophils for the expression of functional NPY receptors and assessed whether NPY modulates the phagocytotic abilities of the neutrophils. 2. Materials and methods 2.1. Samples and peptides For the in vitro experiments with human neutrophils, heparinized blood was collected from healthy volunteers. The institutional ethics committee approved the study and informed consent was obtained from the donors. NPY (Michel et al., 1998; Tatemoto et al., 1982; Tatemoto et al., 1985), pancreatic polypeptide (Michel et al., 1998), Leu31Pro34-NPY (Fuhlendorff et al., 1990), NPY13–36 (Doods et al., 1999; Michel et al., 1998; Schwartz et al., 1987) and D-Trp32 (Hwa et al., 1999) were obtained from PolyPeptide Laboratories (Wolfenbüttel, Germany). BIBO 3304 (Wieland et al., 1998) and BIIE 0246 (Doods et al., 1999) were kindly provided by Boehringer Ingelheim (Biberach, Germany). 2.2. Isolation of neutrophil granulocytes Venous blood samples (10 ml), anticoagulated with Lithiumheparin, were drawn from healthy blood donors in the morning. To obtain neutrophils, blood samples were underlayed with Ficoll-Paque (Amersham Biosciences, Uppsala, Sweden) and centrifuged for 30 min to separate neutrophils from peripheral blood mononuclear cells (PBMC). The granulocyte layer usually
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contained N98% granulocytes with b1% contamination with PBMC and we routinely retrieved 1–3 million granulocytes from an individual preparation that were then used for the assays described below. 2.3. PCR For RT-PCR neutrophils were separated from peripheral blood (40 ml) of healthy volunteers using a cell sorter gating on granulocytes according to the FSC versus SSC properties of the cells (FACStar plus, Becton Dickinson, Heidelberg, Germany). Since NPY receptors are abundantly expressed in the central nervous system, we used commercially available human brain suspensions as positive controls (BD Biosciences, Palo Alto, CA). RNA was extracted using the RNeasy spin column purification kit (Qiagen, Hilden, Germany), and concentrations were measured using a photometer (OD 260 nm). M-MLV reverse transcriptase (Invitrogen, Karlsruhe, Germany) was used to perform reverse transcription. Identical amounts of RNA were used. PCR reactions were performed in a 20 μl volume with 0.5 μM primers: Y1R sense 5′GTAGGTATTGCTGTGATTTGGG-3′ and antisense 5′CTCTGGAAGTTTTTGTTCA GGA-3′; Y2R sense 5′CCTACTGCTCCATCATCTTGC-3′ and antisense 5′GTAGTTGCTGTTCATCCAGCC-3′; Y4R sense 5′GTGTTTCAC AAGGGCACCTA-3′ and antisense 5′ TGCCACTTAGCCTCAGGGA-3′, Y5R (sense) 5′-AGCCATGTGCCATATCATGC-3′ and (anti-sense) 5′-GAGGCAGGATATACTGCACT-3′, NPY sense 5′AGCCATGTGCCATATTAGC3′ and antisense 5′GGCAGAATA TACTGAACTAGC-3′; β-tubulin rRNA sense 5′TTCCCTGG CCAGCTSAANGCNGAGCTNCGCAAG-3′ and antisense 5′-CATGCCCTCG CCNGTGTACCAGTGNANGAAGGC-3′, 1.5 mM MgCl2 and 10 μl Qiagen Mastermix. After 10 min denaturation at 95 °C, 32–38 cycles with 95 °C for 30 s, 54 °C (β-tubulin)/ 58 °C (Y1R, Y5R)/59 °C (Y4R)/61 °C (NPY, CD26) for 30 s, and 72 °C 60 s were performed. After PCR amplification, the reactions were analyzed by gel electrophoresis. Samples were diluted 1:2 with loading dye and loaded onto 2% agarose gels containing ethidium bromide. Gels were run for 45 min at 80 V. Expression of 18S served as positive control. Quantitative analysis of NPY receptor RNA was performed using a smart cycler (Cepheid, France) taking advantage of the QuantiTec SYBR Green™ technology (Qiagen). RNA from sorted neutrophils was isolated as described above. Each PCR cycle comprised melting at 95 °C for 15 s, annealing at a temperature specific for each gene (hCD26: 56 °C, hNPY: 54 °C, hNPYY1: 57.5 °C, hNPYY2: 58 °C, hNPYY4: 55 °C, hNPYY5: 56 °C, hGAPDH: 55 °C) for 30 s, and an extension at 72 °C for 30 s. Each PCR amplification was performed in triplicates. The optimal parameters for the PCR reactions were defined empirically. The purity of the amplified PCR products was verified by melting curves. hGAPDH was used as housekeeping gene. The specificity of the oligonucleotides (see above) used to amplify the NPY receptors was determined using cell lines transfected with specific cDNAs (SK-N-MC cells — Y1R; stably transfected HEC-1B — Y5; transiently transfected COS-7cells — Y2R and Y4R).
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2.4. Radio ligand binding For the radio ligand assay, granulocytes were enriched by Ficoll density gradient as described above. Five to seven million granulocytes were homogenized in 50 volumes of ice-cold Tris–HCl buffer (50 mM Tris, 100 mM NaCl, 5 mM KCl, pH 7.5) and centrifuged for 15 min at 48,000 g (0 °C). The resulting membranes were resuspended in 200 volumes of incubation buffer (50 mM Tris, 100 mM NaCl, 1 mM EGTA, 5 mM MgCl2 supplemented with 50 μM PMSF, 1% BSA, 0.025% Bacitracin and 1 μM phosphoramidon) and stored. The homogenate (0.1 ml) was incubated for 2 h at room temperature in the presence of 30 pM 125I-NPY and increasing concentrations of unlabeled NPY in a total volume of 0.25 ml. The incubation was stopped by centrifugation (10 min, 3000 g), the pellet washed with 0.25 ml of incubation buffer without resuspension, recentrifuged and membrane-bound radioactivity was counted. Non-specific binding was determined in the presence of 100 nM NPY. The binding assays were performed 3 to 6 times, each value in triplicate.
with 2 ml PBS and addition of PI (5 × 10− 5 mol/l) to stain the DNA of cells and bacteria, the samples were analyzed within 20 min by flow cytometry. A linear region on the sharp peak signal in the histogram of FL3 was set to discriminate leukocytes from bacteria. With this, linear region bacteria and cell debris were excluded by live gating during acquisition. In each sample 20,000 events were measured. FSC/SSC dot plots were used to gate neutrophils. The percentage of phagocytizing neutrophils was calculated after setting a quadrant region that was adjusted to the negative control. 2.7. Statistical analysis Data were analyzed using one-factor analyses of variance (ANOVA) for repeated measures and were followed by Fisher LSD post hoc analysis at specific dosages, if appropriate. Results are demonstrated as percentile change from controls. Values represent means ± SEM and asterisks in the figures indicate significant post hoc effects.
2.5. Assessment of respiratory burst Separated neutrophils were resuspended in PBS and incubated with 0.11 mM dihydrorhodamine (DHR, Sigma, Germany) at 37 °C. Various concentrations of NPY and NPYspecific agonists and antagonists were added. After 10 min of incubation the respiratory burst (RB) was initiated using fMLP (0.01 mmol l− 1, Sigma, Seelze, Germany). 10 μl of propidium iodine (PI, Sigma, Seelze, Germany) was included to differentiate between viable and dead cells. Fifteen minutes later, samples were put on ice to terminate the reaction. Cells were analyzed by flow cytometry (Coulter Epics, Krefeld, Germany) using the blue-green excitation light (488 nm argonion laser). Neutrophils were included by setting a polygonal gate in the forward (FSC) versus the sideward scatter signals (SSC). Twenty thousand events were included for each measurement. The DHR emission was filtered and measured with the green photomultiplier (FL1). The percentage of DHR positive cells in the test samples was then determined by counting the number of events above this marker position and dividing it with the whole events observed. PI emission was detected with the red photomultiplier (FL3). Results are expressed as percentile changes compared to unstimulated controls. 2.6. Phagocytosis assay Phagocytosis of opsonized Escherichia coli (Phagotest, Orpegen, Heidelberg, Germany) was assessed using a commercial test kit. Briefly, whole blood samples were incubated with 5 μl fluorescein isothiocyanate (FITC)-labeled opsonized E. coli (1 × 109/ml E. coli, Orpegen, Germany) at 37 °C for a period of 10 min. Negative controls were kept on ice. The cells were treated with a quenching solution to avoid false positive staining of bacteria adhered to the cell surface. After centrifuging and additional washing with PBS (250 g, 5 min, 4 °C), resuspension
Fig. 1. (A) Human neutrophils (PMN) fail to express NPY mRNA, but express mRNA encoding the Y1, Y2, Y4 and the Y5 receptors. For RT-PCR, human neutrophils were freshly isolated from peripheral blood of healthy volunteers using a cell sorter. A commercially available suspension of human brain was used as positive control. β-tubulin served as house keeping gene. One representative experiment out of three is shown. (B) Quantitative analysis of receptor expression before and after stimulation with 10 μM fMLP in comparison to PBMC and positive control levels (brain). PCR was performed with 38 cycles and the PCR products had the following sizes. Y1 receptor: 188 bp; Y2 receptor: 183 bp; Y4 receptor: 190 bp; Y5 receptor: 187. (C) Binding of 125I-labeled NPY to membranes from human neutrophils. IC50 was determined at 78 nM. Neutrophils were homogenized to obtain membrane suspensions. The membranes were incubated with 125I-labeled NPY (30 pM) and various concentrations of unlabeled NPY were added. Binding of 125I-labeled NPY is competed by native NPY, indicating the specificity of the binding. Each concentration was determined in triplicates.
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3. Results 3.1. Human neutrophils express NPY receptors A critical prerequisite for NPY to modulate human neutrophil function is the expression of functional NPY receptors. In a first step we investigated NPY receptor mRNA expression using RTPCR. Though separation of neutrophils by density gradients yields cell suspensions with sufficient purity for functional assays (b3% non-neutrophils), RT-PCR analysis was performed with sorted neutrophils to ensure maximal purity. As indicated in Fig. 1A, we found mRNA encoding the NPY receptor subtypes Y1, Y2, Y4 and Y5 in human neutrophils. Commercially available human brain suspensions were used as positive controls. To extent this analysis, we also performed real time PCR on enriched neutrophils before and after stimulation with fMLP and compared relative expression of the receptors with positive controls and peripheral blood mononuclear cells (PBMC). As shown in Fig. 1B, the expression hierarchy of the receptors followed the following order: Y4 receptor N Y1 and Y2 receptor N Y5 receptor. Whereas Y1, Y4 and Y5 receptor expression remained stable after stimulation with fMLP, Y2 receptor expression was significantly increased after stimulation. Even though the expression levels for the Y1, Y2 and Y4 receptor were comparable between neutrophils and PBMC, we found no relevant Y5 receptor expression in PBMC. In a second step we employed radio ligand binding studies to determine whether specific binding sites, indicative for the presence of NPY receptors, exist on the surface of the neutrophils. In these studies we detected binding of radiolabeled 125 I-NPY to the neutrophil membranes (Fig. 1C). Binding of 125I-NPY to the membranes can be competed by adding increasing concentrations of unlabeled NPY, demonstrating that binding of NPY to the human neutrophils is specific. Thus, based on RT-PCR and radio ligand binding studies, we show here that human neutrophils possess NPY receptors. 3.2. Priming of the fMLP-induced respiratory burst is mediated via the NPY Y5 receptor subtype Given that human neutrophils express NPY receptors we next explored whether these receptors are functional. To this end, we stimulated freshly isolated human neutrophils with E. coli and fMLP in vitro, and determined their production of reactive oxygen species by flow cytometry. NPY significantly increased the respiratory burst induced by fMLP in a dose-dependent manner (Fig. 2A), demonstrating that the NPY receptors on the neutrophils are fully functional. The results from the RT-PCR suggested a role for the Y1, Y2, Y4 and Y5 receptors in the priming of the respiratory burst elicited by NPY. To clarify the functional involvement of these receptor subtypes, we employed an array of different NPY receptor-specific agonists and antagonists. Among the tested agonists, only Leu31Pro34-NPY and D-Trp32 induced a significant intensification of the respiratory burst that was comparable to the action of native NPY (Fig. 2B). With Leu31Pro34-NPY being a selective agonist at the Y1 and the Y5 receptor, and D-Trp32 selectively activating the Y5 receptor,
Fig. 2. A. NPY intensifies the fMLP-induced respiratory burst in a dose-dependent fashion. Neutrophils were isolated from peripheral blood of healthy volunteers and were stimulated with 10 μM fMLP in the presence of various concentrations of NPY. B and C. Pharmacological assessment of the involvement of different NPY receptor subtypes in the fMLP-induced respiratory burst. Neutrophils were isolated from peripheral blood of healthy volunteers and were stimulated in vitro with 10 μM fMLP in the presence of different NPY receptor-specific agonists (B). Only NPY (10− 5 M), Leu31-Pro34-NPY (10− 5 M) and D-Trp32 (10− 5 M) significantly intensified the respiratory burst, suggesting that the effect is mediated by the Y1 and/or the Y5 receptor. Combining NPY with the Y1 receptor antagonist BIBO 3304 (10− 4 M) failed to alter the intensification elicited by NPY (C), demonstrating that Y1 receptor activation plays no significant role in the intensification of the respiratory burst by NPY. The respiratory burst was assessed using a flow cytometer. Values are expressed as relative changes from controls. Results are the mean ± SEM of n = 5 experiments.
these data suggest that the priming of the respiratory burst is mediated via the Y5 receptor subtype. Formally, the intensification of the respiratory burst by Leu31Pro34-NPY could also indicate additional Y1 receptor mediated effects. This possibility, however, is ruled out by the observation that a combination of the selective Y1 receptor antagonist BIBO3304 and native NPY results in a comparable intensification of the respiratory burst as elicited by both native NPY (Fig. 2C) and D-Trp32. Thus, the NPY-induced intensification of the respiratory burst by human neutrophils is mediated via the Y5 receptor subtype. 3.3. Both the Y1 and the Y2 receptor subtype modulate phagocytosis by neutrophils Another hallmark of neutrophil function is their ability to ingest microorganisms and particles. To evaluate whether NPY
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also interferes with this important neutrophil function, we analyzed phagocytosis of fluorescent-labeled E. coli by flow cytometry. Fig. 3A demonstrates a dose-dependent, bimodal
effect of NPY on neutrophil phagocytosis: whereas low concentrations significantly potentiate phagocytosis, increasing dosages eventually inhibit phagocytosis by about 25%. It was interesting to assess whether this effect also depends on Y5 receptor activation or whether other receptor subtypes are involved. Notably, phagocytosis can only be inhibited using Leu31Pro34-NPY and the Y2 receptor-selective agonist NPY13–36 (Fig. 3B), suggesting a combined involvement of the Y1 and the Y2 receptor. Even though the effects of Leu31Pro34-NPY could formally also be attributed to Y5 activation, this is highly unlikely, as the Y5 receptor agonist D-Trp32 had no effect. Again, we reevaluated these effects by combining native NPY with selective antagonists of the Y1 and the Y2 receptor. In the presence of both the Y1 receptor antagonist BIBO3304 and the Y2 receptor antagonist BIIE3246 (Fig. 3C), NPY fails to significantly inhibit phagocytosis. Thus, these findings demonstrate that NPY modulates phagocytosis via combined activation of the Y1 and the Y2 receptor. 4. Discussion
Fig. 3. A. Phagocytosis of fluorescence-labeled E. coli by human neutrophils is modulated by NPY. Neutrophils, isolated from peripheral blood of healthy volunteers, were incubated with FITC-labeled E. coli for 10 min at 37 °C. Cells were washed and binding of labeled E. coli to the cell membrane was avoided using a quenching solution. FITC-labeled E. coli were detected by flow cytometry. B and C. Pharmacological assessment of the involvement of different NPY receptor subtypes in the modulation of phagocytosis. Only NPY (10− 5 M), Leu31-Pro34NPY (10− 5 M) and NPY13–36 (10− 5 M) significantly inhibited phagocytosis, suggesting a combined action via the Y1 and the Y2 receptor (B). As the inhibition could be abrogated by addition of the Y1 receptor antagonist BIBO 3304 (10− 4M) and the Y2 receptor antagonist BIIE3226 (C), it is further substantiated that the ability of NPY to modulate phagocytosis is mediated via a combined interplay at the Y1 and the Y2 receptor subtype. Values are expressed as relative changes from controls. Results are the mean ± SEM of n = 5 experiments.
In the present study we have demonstrated that human neutrophils possess functional NPY receptors. Importantly, neutrophil function is regulated by NPY in a complex manner with different NPY receptor subtypes responsible for particular functions. Activation of the Y5 receptor potentiates the production of reactive oxygen species elicited by fMLP and parallel Y1 and Y2 receptor stimulation modifies phagocytosis of E. coli. A critical prerequisite for leukocytes to respond to signals from the nervous system is the expression of specific receptors for neurotransmitters or neuropeptides on their surfaces. Therefore, a major aim of the present study was to determine whether NPY receptors are present on human neutrophils. Our qualitative and quantitative PCR results revealed the presence of mRNA encoding for the Y1, Y2, Y4 and Y5 receptors in human neutrophils and established differential expression patterns for the respective receptors. Notably, when compared to a commercially available human brain suspension, expression levels of all NPY receptors were higher in neutrophils. As mRNA expression does not necessarily imply that a functional receptor protein is also present on the cell surface, we additionally employed radio ligand studies to investigate whether specific binding sites for NPY are present on neutrophils. 125 I-labeled NPY bound to membrane suspensions prepared from neutrophils and these binding sites were shown to be specific, as increasing amounts of unlabeled NPY displaced radio-labeled NPY in a dose-dependent manner. The competition curve followed a classical sigmoid curve and the IC50 was determined as 78 nM. Given this 100-fold higher IC50 value of the neutrophils compared to transfected cell lines (Fabry et al., 2000), it appears that the NPY receptors are not very densely distributed on neutrophils. Low-level expression also explains as to why other less sensitive methods of tracking NPY receptors expression with radio ligands (3H) produced inconclusive results (data not shown). Nevertheless, based on the presence of NPY receptor mRNA in neutrophils and the
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results from the binding study, we conclude that human neutrophils express NPY receptors. To assess the functional capacity of the expressed NPY receptors we employed two different in vitro assays. The production of reactive oxygen species and the phagocytosis of invading microbes are essential for the neutrophil to defend the body against invading microbes (Aldridge, 2002; Quinn and Gauss, 2004). Therefore, we tested the effect of NPY on the respiratory burst induced by fMLP stimulation in vitro and found that NPY potentiates this critical neutrophil function. Notably, not only the respiratory burst is significantly altered by NPY, also phagocytosis of fluorescence-labeled E. coli is subject to significant modulation by NPY. As both of the effects elicited by NPY follow a clear dose-dependency, the results prove that the detected NPY receptors on human neutrophils are functionally active. Despite the demonstration of NPY receptor mRNA, NPYspecific binding sites and functional in vitro effects of NPY on human neutrophils, the findings discussed so far fail to elucidate whether all or only some of the NPY receptor subtypes detected in PCR are actually involved in these effects. Therefore, we employed an array of different NPY-specific agonists and antagonists to determine pharmacologically which of the NPY receptor subtypes mediate the functional effects of NPY on the respiratory burst and phagocytosis. The intensification of the respiratory burst by NPY can be mimicked by the Y1 and Y5 receptor-specific agonist Leu31-Pro34-NPY and the Y5 receptorselective peptide D-Trp32, suggesting that the Y5 receptor mediates this intensification. Though Leu31-Pro34-NPY also exerts agonistic activities at the Y1 receptor, the possibility of an additional involvement of the Y1 receptor is excluded by our finding that NPY still potentiates the respiratory burst when the Y1 receptor is pharmacologically blocked by BIBO 3304. The pharmacological data, therefore, demonstrates that NPY potentiates the respiratory burst via activation of the Y5 receptor. What makes this finding particularly interesting is the observation that the expression levels for the Y5 receptor are rather low when compared to the other receptors, suggesting that this receptor is very efficient at modulating one of the most critical functions of neutrophils. Using a similar pharmacological approach, we also analyzed which receptor subtypes mediated the effects of NPY on phagocytosis. Interestingly, the results revealed a different pattern as both Leu31-Pro34-NPY and the Y2 receptor-selective agonist NPY13–36 mimic the inhibition of phagocytosis. Again we also used the combination of NPY and Y1 and Y2 receptor antagonists to further evaluate the role of these receptors in phagocytosis. Under both conditions the inhibition of phagocytosis by NPY is abrogated, indicating that both the Y1 and the Y2 receptor subtype mediated this effect. This finding in combination with the absence of any effects of D-Trp32 also argues against a role for the Y5 receptor that could formally be implicated by the action of Leu31-Pro34-NPY. Thus, the action of NPY on phagocytosis involves activation of the Y1 and the Y2 receptor. A more precise look at the data reveals some interesting additional details. Even though the Y1/Y5 receptor agonist Leu31-Pro34-NPY inhibits phagocytosis as potently as
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NPY, the combination of NPY with either a Y1 or a Y2 receptor antagonist — which should result in the stimulation of the Y1 and the Y2 receptor respectively — failed to significantly inhibit phagocytosis. To explain this apparent contradiction, it has to be taken into consideration that even though Leu31-Pro34NPY and NPY13–36 act as potent selective agonists at their respective receptors (Doods et al., 1999; Fuhlendorff et al., 1990), they still have the ability to activate other NPY receptors with a much lower affinity (Michel et al., 1998). Thus, in the situation where either Leu31-Pro34-NPY or NPY13–36 was added alone, they also exhibited a partial — though weak — agonism at the Y2 or Y1 receptor respectively. Consistent with this notion, addition of NPY in the presence of a Y1 or Y2 antagonist, where a partial agonism at the other receptor is ruled out, proved inefficient at inhibiting phagocytosis. This suggests that both the Y1 and Y2 receptor have to be activated in parallel for NPY to exert its full inhibitory activity on phagocytosis. In the light of a potent upregulation of the Y2 receptor upon stimulation (Fig. 1B) and the fact that the blockade of the Y1 receptor was not quite as efficient in restoring phagocytosis (Fig. 3C), it appears that the Y2 receptor delivers the stronger signal during this interplay. It will be exciting for future studies to unravel the molecular mechanism underlying this complex interaction. Even though the Y4 receptor was detected on the mRNA level and in comparison to the other receptor, was expressed at the highest level, the absence of any functional effect of PP, which preferentially activates the Y4 receptor, argues against any functional effects of the Y4 receptor protein on phagocytosis and the respiratory burst of human neutrophils. Verifying these findings by detecting the very receptor proteins on the surface of the neutrophils by flow cytometry, for example, is at present not possible due to the absence of monoclonal antibodies for NPY receptors. We are currently undertaking attempts to generate such antibodies. Taken together, the PCR results, the radio ligand binding studies and the in vitro effects demonstrate that human neutrophils express fully functional Y1, Y2 and Y5 receptor subtypes. Formally, this study cannot prove that the observed receptor-specific effects of NPY on differential neutrophil function are entirely the consequence of a direct interaction between NPY and neutrophils, as the methodological approach of using fresh human neutrophils always has the limitation that some contaminating blood cells are present in the preparations. Given that receptor expression levels on the neutrophils were either similar (for the Y1, Y2 and Y4 receptor) or higher (for the Y5 receptor) when compared to PBMC, indicates that the NPY receptors are expressed on a higher level in neutrophils than in the heterogeneous PBMC population, pointing towards direct rather than indirect effects. Further studies are necessary to absolutely exclude indirect effects. These studies extend our previous findings demonstrating the presence of functional Y1 receptors on rat PBMC (Bedoui et al., 2002a,b) and mouse T lymphocytes (Bedoui et al., 2003b; Petitto et al., 1994). With previous reports showing that human lymphocytes and monocytes produce NPY (Schwarz et al., 1994) on the one hand and the presence of functional NPY receptors on human neutrophils on the other, it was possible that human neutrophils
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also produce NPY, which in turn might be involved in an autocrine feedback loop. However, we found no support in favor of this hypothesis, as neither NPY mRNA was detectable in resting human neutrophils as well after 24 h of in vitro stimulation with fMLP, nor did the Y1 and Y2 receptor antagonists exert any effect on the neutrophils in vitro (data not shown). So, we are left with a complex picture that very much depends on which receptor is activated and at what concentration NPY is present. In low doses, NPY increases the phagocytosis of E. coli without changing the respiratory burst. However, in higher concentration NPY not only inhibits phagocytosis, but also augments the respiratory burst. Thus, it appears that NPY has the ability to switch the way a neutrophil deals with invading pathogens. When NPY is present at low doses, the pathogen is taken up and intracellular mechanisms are in charge of its elimination. Conversely, in microenvironments of higher NPY concentrations, the neutrophil allows fewer pathogens to be ingested and rather upregulates its ability to destroy the pathogen by extracellular means. The latter way of action might be particularly important in situations where large numbers of pathogens are encountered, ensuring that other cells, such as professional antigen presenting cells, can access the pathogen for antigen display to branches of the adaptive immune system. These findings also have interesting clinical implications. Septicaemia, during which the uncontrolled dissemination of pathogens and their products severely compromises the function of the immune system, still poses a major clinical problem and is associated with high mortality (Sessler et al., 2004). Over the last years, it has been conclusively shown that, in fact, the sympathetic nervous system also plays an important role during septicaemia through its effects on the immune system. Whereas this action has been traditionally attributed to the function of catecholamines (Kohm and Sanders, 2001), several lines of evidence also indicate that NPY can exert such protective-like effects during experimental septicaemia (Hauser et al., 1993; Nave et al., 2004). Given that patients with septicaemia often develop a functional resistance to exogenous catecholamines, the findings outlined in this study, might provide a first step towards the clinical utilization of NPY in patients with septicaemia in the future. Acknowledgements The excellent technical assistance of S. Kuhlmann and G. Peper, and the correction of the English by S. Fryk are very much appreciated. The authors would also like to thank Dr. R. Pabst for critical review of the manuscript. References Aldridge, A.J., 2002. Role of the neutrophil in septic shock and the adult respiratory distress syndrome. Eur. J. Surg. 168, 204–214. Bedoui, S., Kuhlmann, S., Nave, H., Drube, J., Pabst, R., von Hörsten, S., 2001. Differential effects of neuropeptide Y (NPY) on leukocyte subsets in the blood: mobilization of B-1-like B-lymphocytes and activated monocytes. J. Neuroimmunol. 117, 125–132. Bedoui, S., Lechner, S., Gebhardt, T., Nave, H., Beck-Sickinger, A.G., Straub, R.H., Pabst, R., von Hörsten, S., 2002a. NPY modulates epinephrine-induced
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