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Neuropeptide Y modulates functions of inflammatory cells in the rat: Distinct role for Y1, Y2 and Y5 receptors
Peptides 32 (2011) 1626–1633
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Neuropeptide Y modulates functions of inflammatory cells in the rat: Distinct role for Y1, Y2 and Y5 receptors Katarina Mitic´ a , Stanislava Stanojevic´ a , Nataˇsa Kuˇstrimovic´ a , Vesna Vujic´ b , Mirjana Dimitrijevic´ a,∗ a b
Institute of Virology, Vaccines and Sera, “Torlak”, Immunology Research Center “Branislav Jankovi´c”, Vojvode Stepe 458, 11152 Belgrade, Serbia Institute of Chemistry, School of Medicine, University of Belgrade, Belgrade, Serbia
a r t i c l e
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Article history: Received 21 April 2011 Received in revised form 7 June 2011 Accepted 7 June 2011 Available online 14 June 2011 Keywords: Granulocytes Inflammation Monocytes Neuropeptide Y Rat Y receptor subtypes
a b s t r a c t Neuropeptide Y (NPY) has been reported to be a potent anti-inflammatory peptide with ability to directly modulate activity of granulocytes and macrophages. The present study aimed to correlate the effects of NPY in vivo on lipopolysaccharide-induced air-pouch exudates cells and in vitro on peripheral blood leukocytes functions. The role of different Y receptors was examined using NPY-related peptides and antagonists with diverse subtype specificity and selectivity for Y receptors. Y1, Y2 and Y5 receptors were detected on air-pouch exudates cells (flow cytometry) and peripheral blood granulocytes (immunocitochemistry). NPY in vivo reduced inflammatory cells accumulation into the air pouch, and decreased their adherence and phagocytic capacity via Y2/Y5 and Y1/Y2 receptors, respectively. Quite the opposite, NPY in vitro potentiated adhesiveness and phagocytosis of peripheral blood granulocytes and monocytes by activating Y1 receptor. The differences between in vivo and in vitro effects of NPY on rat inflammatory cells functions are mostly due to dipeptidyl peptidase 4 activity. In addition, suppressive effect of NPY in vivo is highly dependent on the local microenvironment, peptide truncation and specific Y receptors interplay. © 2011 Elsevier Inc. All rights reserved.
1. Introduction Neuroendocrine derived factors in compliance with mediators originated from leukocytes and macrophages participate in the development and resolution of acute inflammation. Neuropeptides are responsible for the transmission of pain signals, regulation of vascular tonus and permeability, accumulation of inflammatory cells and stimulation of secretion by immune and endocrine cells [27,29]. The finding that inflammatory cells are located in the vicinity of nerve endings containing neuropeptides implicated that neuropeptides directly affect the functions of these cells [30]. Neuropeptide Y (NPY) is found in the sympathetic nerve endings and released upon sympathetic nervous system activation along with norepinephrine [12]. Over the last decade, an increasing number of evidences point to the involvement of NPY in the modulation of inflammatory reaction [8,11,15,20,34]. It has been shown that NPY in vitro enhanced
Abbreviations: NPY, neuropeptide Y; LP-NPY, [Leu31,Pro34]-NPY; hAAib-NPY, [hPP1-17, Ala31, Aib32]-NPY; LPS, lipopolysaccharide; DP4, dipeptidyl peptidase 4; NBT, nitro blue tetrazolium chloride; PBS, phosphate buffered saline; MEM, minimal essential medium without phenol red; MPO, myeloperoxidase; ODs, optical densities. ∗ Corresponding author. Tel.: +381 11 3976 674; fax: +381 11 2471 838. ´ E-mail address: [email protected] (M. Dimitrijevic). 0196-9781/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2011.06.007
functions of inflammatory cells such as adherence [6,25] chemotaxis [6,31], cytokine secretion [16,35] and production of reactive oxygen metabolites [2,6,10] and nitric oxide [7,8]. Some inhibitory effects of NPY on inflammatory cells activity, like reduction of granulocyte oxidative burst [7] and phagocytosis [2,7] have also been reported. NPY exerts biological activity via all subtypes of Y receptors, which are the Y1-5 receptor subtypes, while the y6 receptor has not been identified in rats [13]. An unusual feature of Y receptor family is the lack of sequence identity, particularly between the Y1, Y2 and Y5 receptor subtypes, which share only 30% sequence homology between themselves [24]. In addition to tissue-specific receptor subtype expression, NPY actions may be regulated by its processing by dipeptidyl peptidase 4 (DP4, also known as CD26). DP4 enzyme cleaves NPY1-36 to an Y2/5 receptor preferring peptide NPY3-36, therefore terminating its function on Y1 receptor [23]. A fine regulation of inflammatory cell activity by NPY is achieved through the engagement of different Y receptor subtypes mediating the opposing effects on a specific function, or simply by reducing the effect achieved through the activation of another receptor subtype [7,10]. Furthermore, NPY and/or Y receptors have been related to a number of inflammatory and immune mediated disorders [4,14,15,17,21,28]. Considering many conditions in which levels of NPY might be increased, it is of a great importance to discern its influence on immune and inflammatory cells residing in different compartments. The present study investigated the effect of NPY
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Fig. 1. Characterization of air-pouch exudates cells by flow cytometry: (a) percentage of cells expressing HIS48 and CD11b antigens; (b) representative dot plot showing the expression of HIS48 and CD11b antigens; (c) percentage of cells expressing Y1, Y2 and Y5 receptors; (d) mean fluorescence intensity (MFI) for Y1, Y2 and Y5 receptors; and (e) representative histogram plots of Y1, Y2 and Y5 receptors expression (black line) with negative control samples (omitted primary antibody, gray line). Statistically significant difference (ANOVA): *p < 0.05, MFI for Y5 receptor vs. Y1 and Y2 receptor.
Fig. 2. Immunocytochemical staining of rat peripheral blood granulocytes using anti-Y1-receptor, anti-Y2-receptor, anti-Y5-receptor and anti-CD11b molecule antibodies: (a) Y1-receptor positive cells (brown); (b) Y2-receptor positive cells (brown); (c) Y5-receptor positive cells (brown); (d) CD11b molecule positive cells (light red). Original magnification – ×40. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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in vivo on lipopolysaccharide (LPS)-induced air-pouch exudates cells recruitment and their adherence and phagocytic capacity. LPS has been shown to induce rapid neutrophils infiltration at the inflammatory site [32] resulting in neutralization and/or elimination of Gram negative bacteria. Receptor specificity was examined using NPY-related peptides with different pharmacological specificity for Y receptor subtypes. Also, we have tested the effects of NPY and NPY-related peptides in vitro on activity of peripheral blood monocytes and granulocytes. The involvement of different Y receptor subtypes in NPY-induced modulation of monocyte and granulocyte activity has been further estimated following combined treatment of cells with NPY and selective antagonists of the Y1, Y2 and Y5 receptors.
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2.3. Air-pouch induction and cell harvesting Air-pouches were created by three subcutaneous injections of sterile air into the dorsal region over the period of 7 days. On day 8, rats received one injection into the air pouch containing 10 g of peptide (NPY, Y1,2,4,5 receptor agonist; LP-NPY, Y1,5 receptor agonist; NPY3-36, Y2,5 receptor agonist; NPY13-36, Y2 receptor agonist; or hAAib-NPY, Y5 receptor agonist) and 20 g LPS in 2 ml of PBS. Control rats were injected with 20 g LPS in 2 ml of PBS. Cells were obtained by air-pouch lavage with 10 ml of PBS, 3 h later, washed three times in PBS, and adjusted to 2.5 × 106 or 1 × 107 cells/ml with regard to the assay. 2.4. Isolation of monocytes and granulocytes from peripheral blood Rats were bled by cardiac puncture in isoflurane anesthesia and euthanized immediately after. Briefly, heparinized blood was mixed with Dextran T500 (6% w/v in PBS) in order to obtain leukocyte enriched plasma. Mononuclear cells and granulocytes were separated by density gradient centrifugation over Percoll
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Human/rat NPY (Y1,2,4,5 receptor agonist), [Leu31,Pro34]-NPY (LP-NPY, Y1,5 receptor agonist), NPY3-36 (Y2,5 receptor agonist) and NPY13-36 (Y2 receptor agonist) were obtained from Polypeptide Laboratories, Wolfenbuttel, Germany. The Y5 receptor agonist [hPP1-17, Ala31 , Aib32 ]-NPY (hAAib-NPY) was a gift from Prof. Annette G. Beck-Sickinger, University of Leipzig, Germany. BIBO3304 (Y1 receptor antagonist), and BIIE0246 (Y2 receptor antagonist) were kindly provided by Dr. Henry Doods, Boehringer Ingelheim, Biberach, Germany. L152804, Y5 receptor antagonist, was obtained from Tocris (Bristol, UK). Lipopolysaccharide (LPS from E. coli) and zymosan were purchased from Sigma (St. Louis, MO, USA). Nitro blue tetrazolium chloride (NBT) was obtained from Serva (Heidelberg, Germany). Phosphate buffered saline (PBS) and Minimal essential medium without phenol red (MEM) were obtained from Institute “Torlak”, Belgrade, Serbia.
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Female Dark Agouti rats, 4 months of age were obtained from the breeding colony at Immunology Research Center “Branislav ´ Belgrade (Serbia). The animals were housed individuJankovic”, ally in standard macrolone cages with free access to food pellets and tap water. All procedures involving the animals and their care were approved by our Institutional Animal Care and Use Committee and followed principles described in the European Community’s Council Directive of 24 November 1986 (86/609/EEC).
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Fig. 3. The effects of direct injection of NPY, LP-NPY, NPY3-36, NPY13-36 and hAAibNPY (10 g/20 g LPS/pouch) into the air pouch on: (a) total cell recruitment; (b) adherence capacity of cells stimulated with 1 g/ml of LPS; and (c) phagocytic capacity of cells stimulated with 125 g/ml of zymosan. The number of rats per group was eight. Statistically significant differences (t-test): *p < 0.05, and # p < 0.001, vs. control.
( = 1.077 g/ml). Granulocytes were isolated from pellets after lysis of erythrocytes with isotonic solution of NH4 Cl. 2.5. Flow cytometry For immunophenotyping, 1 × 106 air-pouch exudates cells per sample were washed and resuspended in flow cytometry buffer (PBS supplemented with 0.09% NaN3 ). All subsequent incubations were performed at +4 ◦ C in the dark, 30 min in duration, and were followed by through washings. Double staining of cells was performed using the fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-conjugated antibodies. The cells were directly stained with previously titrated mouse monoclonal anti granulocyte (HIS48) IgM-PE, anti-rat CD11b IgG-FITC (clone ED8) and anti-rat CD26 IgG-PE (clone Ox61). The cells were kept at +4 ◦ C in the dark prior to the analysis.
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Fig. 4. The in vitro effects of NPY, LP-NPY, NPY3-36 and hAAib-NPY at 10 M concentration on adherence capacity of peripheral blood granulocytes (a) and monocytes (b) stimulated with 1 g/ml of LPS. The number of cell samples per treatment was eight. Statistically significant differences (t-test): *p < 0.05, **p < 0.01, and # p < 0.001, vs. control.
For detection of Y receptors, cells were stained with polyclonal goat-anti Y receptor IgG (Y1 receptor, D-16; Y2 receptor, L-17; and Y5 receptor, N-20) followed by FITC-conjugated rabbit antigoat antibody. For the intracellular antigen immunolabeling, the cells were fixed with 0.25% paraformaldehide and permeabilized by 0.2% Tween 20 (15 min, +4 ◦ C) prior to the addition of antirat Y1 and Y2 antibodies, followed by FITC-conjugated anti-goat antibody as a secondary reagent. Antibodies were purchased from Becton Dickinson, San Jose CA, USA (CD11b-FITC, CD26-PE), Santa Cruz Biotechnology, Santa Cruz CA, USA (HIS48-PE and Y1, Y2 and Y5 receptors), and Sigma–Aldrich Chemie, Taufkirchen, Germany (rabbit anti-goat IgG-FITC). After extensive washing in flow cytometry buffer, all samples were analyzed on a FACScan flow cytometer using CellQuest software (Becton Dickinson, Mountain View, CA, USA). In addition, Y1, Y2 and Y5 receptor expression was analyzed by estimation of the mean fluorescence intensity (MFI), which correlates, in a relative sense, to the number of molecules expressed by the cell [18].
N-20) and mouse anti-rat CD11b IgG. The LSAB+ system was used to detect anti-rat Y receptor antibodies binding, while the EnVision G|2 Doublestain System was used to visualize antirat CD11b receptor antibody binding. After endogenous alkaline phosphatase and peroxidase activity was quenched, cells were washed in PBS and subsequently incubated with anti-rat primary antibodies diluted in background reducing antibody diluent (DakoCytomation) overnight at 4 ◦ C. Binding of the primary antibody was detected using appropriate secondary reagents and either 3,3 -diaminobenzidine (DAB+) (LSAB+ kit) or permanent red as chromogen (EnVision kit). Finally, cytospins were hematoxylin counterstained and cover-slipped. To verify staining specificity, the primary antibodies were substituted with antibody diluent. No specific immunoreactivity was detected in these cytospin preparations. A BH2 research microscope (Olympus Optical Co., Ltd., Tokyo, Japan) equipped with a ColorView III digital camera (Olympus) and Analysis Docu software (Olympus) was used to acquire images.
2.6. Immunocytochemistry
2.7. Adherence assay
Aliquots (105 cells) of peritoneal exudates cells were cytospun onto slides, fixed in ice-cold acetone and then frozen at −20 ◦ C. Prior to immunostaining, they were thawed, washed in phosphate buffered saline (PBS) and when necessary permeabilized with 0.5% saponin in PBS. For visualization of antibody binding LSAB+ and EnVison G|2 Doublestain (DakoCytomation, Glostrup, Denmark) kits were used according to the manufacturer’s instructions. The cells were stained with polyclonal goat-anti Y receptor IgG (Y1 receptor, D-16; Y2 receptor, L-17; and Y5 receptor,
The assay for adherence capacity was based on the measurement of myeloperoxidase (MPO) activity of cells adhered to tissue culture plates [5]. Briefly, individual air-pouch cell suspensions were adjusted to 2.5 × 106 cells/ml and plated at 50 l/well in 96well flat-bottomed tissue culture plates (NUNC, Roskilde, Denmark) previously filled with 50 l/well of 2 g/ml of LPS in MEM. Plates were incubated 60 min at 37 ◦ C in 95% air–5% CO2 , and nonadherent cells were removed by washing the wells twice with MEM. MPO activity was determined using ortho-phenylen-diamine in cit-
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Fig. 5. The effects of BIBO3304 (antagonist of Y1 receptor, 10−6 M), BIIE0246 (antagonist of Y2 receptor, 10−6 M) and L152804 (antagonist of Y5 receptor, 10−6 M) on NPY (10−6 M)-induced increase of adherence capacity of peripheral blood granulocytes (a) and monocytes (b) stimulated with 1 g/ml LPS. The number of cell samples per group was eight. Statistically significant differences (one-factor ANOVA for repeated measures followed by Fisher’s PLSD test; factor: treatment): *p < 0.05, **p < 0.01, # p < 0.001, treatment with specific antagonist (BIBO3304, BIIE0246 or L152804) or NPY vs. none; and a p < 0.05, treatment with NPY and specific antagonist (BIBO3304, BIIE0246 or L152804) vs. treatment with NPY.
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Fig. 6. The in vitro effects of NPY, LP-NPY, NPY3-36 and hAAib-NPY at 10 M concentration on phagocytic capacity of peripheral blood granulocytes (a) and monocytes (b) stimulated with 125 g/ml of zymosan. The number of cell samples per treatment was eight. Statistically significant differences (t-test): *p < 0.05, and **p < 0.01, vs. control.
ric buffer activated with 12% H2 O2 and Triton X-100. The reaction was stopped with 2 M H2 SO4 solution. Optical densities (ODs) were determined on Multiscan Ascent (Labsystems) at 492/620 nm. The results are expressed as OD (492/620 nm) × 1000. Adherence capacity of peripheral blood granulocytes and monocytes was determined in the same manner, except that tissue culture plates were previously filled with 50 l/well of: peptides (NPY, LP-NPY, NPY3-36, hAAib-NPY); specific antagonists of Y receptors (BIBO3304, BIIE0246, L152804); or NPY combined with specific antagonists of Y receptors (NPY/BIBO3304, NPY/BIIE0246, NPY/L152804) in 2 g/ml of LPS in MEM. Drugs were added at final concentration of 1 × 10−6 M. 2.8. Phagocytosis assay The phagocytic capacity was indirectly determined by measuring oxidative burst commenced after engulfment of foreign particles. Specifically, the assay was based on the reduction of yellow NBT to blue formazan by superoxide anion released following phagocytosis of zymozan [26]. Therefore, the magnitude of NBT reduction was proportional to the amount of zymosan particles phagocyted by cells. Individual air-pouch cells suspensions were adjusted to 2.5 × 106 cell/ml, and plated at 100 l/well in 96-well flat-bottomed tissue culture plates. Following incubation for 2 h at 37 ◦ C in 95% air–5% CO2 nonadherent cells were removed by washing the wells twice with MEM. Adherent cells were stimulated with 125 g/ml of zymosan in the presence of 0.5 mg/ml of NBT for 30 min at 37 ◦ C in 95% air–5% CO2 . Cells were fixed with methanol, and ODs were determined on Multiscan Ascent (Labsystems) at 545 nm. The results are expressed as OD (545 nm) × 1000.
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2.9. Statistical analysis Data were analyzed by one-way ANOVA, followed by Fisher’s PLSD test and Student’s t-test. Differences are regarded as statistically significant if p < 0.05. Results are presented as mean ± SE. The statistics were performed using statistical package SPSS10.0 for Windows. 3. Results 3.1. Characterization of air-pouch exudates cells Since the leukocyte integrin CD11b/CD18 (Mac-1; CR3) participates in different phases of inflammation, including the adherence and phagocytosis, the expression of both CD11b and granulocyte marker HIS48 on air-pouch exudates cells were evaluated (Fig. 1). Double staining of cells with HIS48 and CD11b antibodies confirmed that air-pouch exudates cells population elicited with LPS generally consisted of granulocytes (HIS48+CD11b+; 81.0 ± 1.1%) and monocytes (HIS48-CD11b+; 16.2 ± 2.1%), whereas approxi-
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Phagocytic capacity of peripheral blood granulocytes and monocytes was determined in the same manner, except that adherent cells were treated with 100 l/well of: peptides (NPY, LP-NPY, NPY3-36, hAAib-NPY); specific antagonists of Y receptors (BIBO3304, BIIE0246, L152804); or NPY combined with specific antagonists of Y receptors (NPY/BIBO3304, NPY/BIIE0246, NPY/L152804) in 125 g/ml of zymozan. Peptides were added at concentration of 10−10 M, and antagonists of Y receptors at 100 time higher concentration, i.e. 10−8 M.
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Fig. 7. The effects of BIBO3304 (antagonist of Y1 receptor, 10 M), BIIE0246 (antagonist of Y2 receptor, 10 M) and L152804 (antagonist of Y5 receptor, 10−8 M) on NPY (10−10 M)-induced increase of phagocytic capacity of peripheral blood granulocytes (a) and monocytes (b) stimulated with 125 g/ml of zymosan. The number of cell samples per group was eight. Statistically significant differences (one-factor ANOVA for repeated measures followed by Fisher’s PLSD test; factor: treatment): *p < 0.05, **p < 0.01, # p < 0.001, treatment with specific antagonist (BIBO3304, BIIE0246 or L152804) or NPY vs. none; and a p < 0.05, treatment with NPY and specific antagonist (BIBO3304, BIIE0246 or L152804) vs. treatment with NPY.
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mately 3% of HIS48-CD11b-cells represented lymphocytes (Fig. 1a). Representative dot plot of double staining of cells with HIS48-PE and CD11b-FITC is shown in Fig. 1b. Given that activated T lymphocytes express membrane CD26, i.e. DP4 enzyme which is involved in the regulation of NPY biological activity, the presence of CD26+ cells among air-pouch exudates cells was tested by single staining with CD26 antibody. Analysis revealed that less than 3% of air-pouch exudates cells were CD26+ (data not shown). Single staining with antibodies specific for different Y receptor subtypes revealed that Y1, Y2 and Y5 receptors were expressed on roughly 5–8% of air-pouch exudates cells, Fig. 1c. Although there were no differences in the percentage of cells expressing different Y receptor subtypes, mean fluorescence intensity (MFI) for Y5 receptor was significantly lower in comparison with MFI for Y1 and Y2 receptors (Fig. 1d). Representative dot plots of double staining of cells with HIS48-PE and antibodies specific for different Y receptor subtypes are given in Fig. 1e. Immunocytochemical staining of rat peripheral blood granulocytes showed cells expressing Y1, Y2 and Y5 receptors as well as CD11b molecule (Fig. 2). As Y1 and Y2 antibodies recognized intracellular receptor domain in the vicinity of C-terminus, Fig. 2a and b illustrated staining of the inner cell membrane with Y1 and Y2 receptor antibody, respectively. In addition, Fig. 3b represented membrane staining with Y5 receptor antibody that was raised against an extracellular domain of the Y5 receptor. Membrane staining of cells with CD11b antibody was shown in Fig. 2d.
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accompanied with suppressive effect of antagonist per se on cells adherence capacity.
3.4. The effect of NPY and NPY-related peptides in vitro on phagocytic capacity of peripheral blood granulocytes and monocytes NPY (Y1,2,4,5 receptor agonist) and LP-NPY (Y1,5 receptor agonist) at concentration of 10−10 M significantly increased phagocytosis of zymosan in both granulocytes (Fig. 6a) and monocytes (Fig. 6b) from peripheral blood. In contrast, NPY3-36 (Y2,5 receptor agonist) reduced phagocytic capacity of monocytes. Y5 receptor agonist, hAAib-NPY, did not modulate phagocytosis of zymosan, neither in monocytes nor in granulocytes. These findings suggested the role for Y1 receptors in NPY-induced potentiation of phagocytosis in peripheral blood cells. However, both BIBO3304 (Y1 receptor antagonist, 10−8 M) and L152804 (Y5 receptor antagonist, 10−8 M) antagonized NPY-induced increase of phagocytic capacity of granulocytes (Fig. 7a) and monocytes (Fig. 7b). Also, BIBO3304 and L152804 itself, had suppressive effects on zymosan phagocytosis in peripheral blood granulocytes (Fig. 7a). On the other hand, BIBO3304 did not affect, whereas L152804 increased phagocytic capacity of monocytes (Fig. 7b).
4. Discussion 3.2. The effect of NPY and NPY-related peptides in vivo on inflammatory response to LPS NPY (Y1,2,4,5 receptor agonist, 10 g), directly injected into the air-pouch notably decreased LPS-induced cell recruitment, while the same dose of different NPY-related peptides had no effect on the migration of inflammatory cells (Fig. 3a). Finding that LP-NPY (Y1,5 receptor agonist) and hAAib-NPY (Y5 receptor agonist) slightly decrease and increase cells accumulation, respectively, suggested opposite function for Y1 and Y5 receptors regarding cells recruitment into the air pouch. Additionally, ineffectiveness of NPY3-36 (Y2,5 receptor agonist) and NPY13-36 (Y2 receptor agonist) in the modulation of inflammatory cells recruitment discarded the contribution of Y2 receptors. However, NPY and all NPY-related peptides used in the study decreased adherence capacity of inflammatory cells (Fig. 3b). The most pronounced inhibitory effect on adherence capacity of inflammatory cells from air pouch was observed following treatment with NPY3-36 (Y2,5 receptor agonist). Besides, suppressive effect on phagocytic function of air-pouch cells was detected after treatment with NPY and NPY-related peptides with exception for Y5 receptor agonist hAAib-NPY (Fig. 3c). 3.3. The effect of NPY and NPY-related peptides in vitro on adherence capacity of peripheral blood granulocytes and monocytes NPY, at concentration of 10−6 M, in vitro potentiated adherence capacity of LPS-stimulated peripheral blood granulocytes (Fig. 4a), and monocytes (Fig. 4b). Conversely, NPY3-36 (Y2,5 receptor agonist) and hAAib-NPY (Y5 receptor agonist) markedly reduced adherence capacity of granulocytes (Fig. 4a) whereas LP-NPY (Y1,5 receptor agonist) and hAAib (Y5 receptor agonist) decreased adherence of monocytes (Fig. 4b). Furthermore, BIBO3304, BIE0246 and L152804, specific antagonists of Y1, Y2 and Y5 receptors, respectively, antagonized NPY-induced increase of granulocyte adherence capacity (Fig. 5a). NPY-induced increase of monocyte adherence capacity was antagonized by BIE0246 and L152804 (Fig. 5b). It should be noticed that antagonistic action of pharmacological concentrations of BIBO3304, BIE0246 and L152804 (10−6 M) was
Reduced accumulation of inflammatory cells along with their decreased adherence and phagocytic capacity, caused by the in vivo administration of NPY into the inflamed tissue, sustained previously reported anti-inflammatory action of NPY [7–9,33]. Since air-pouch exudates cells mainly consisted of granulocytes, and small but significant proportion of granulocytes expressed Y1, Y2 and Y5 receptors, there was a possibility that NPY affected cells migration through Y receptor-specific mechanism. In view of different Y receptor specificity of LP-NPY (Y1,5 agonist), NPY3-36 (Y2,5 agonist), NPY13-36 (Y2 agonist) and hAAib-NPY (Y5 agonist) the finding that none of them affected cells infiltration in the air pouch advised that NPY suppressed cells accumulation by concomitant activation of Y1 and Y2 receptors. Y1 receptors on peripheral blood mononuclear cells mediated the inhibition of leukocyte mobilization following intravenous NPY in the rat [3]. Nevertheless, earlier studies showed that the effect of NPY on cells migration was dependant on the cell type. Particularly, NPY in vitro enhanced migration of leukocytes [6,22] and suppressed it in monocyte/macrophage cell line Raw 264.7 [1], but itself served as a chemoattractant for human monocytes [31]. In the present study NPY in vitro increased cell adherence via Y1 receptor but peptides devoid of Y1 receptor activity decreased it. Results viewed as a whole suggested that Y1 and Y2/Y5 receptors mediated potentiation and suppression of granulocyte adherence capacity, respectively. In contrast, it has been reported that NPYinduced rise in adherence capacity of rat monocytes [25] and human T lymphocytes [19] was mediated via Y2 receptors. The absence of Y1 receptor-mediated increase of inflammatory cells adhesion following in vivo treatment with NPY may have originated from DP4-induced conversion of NPY to NPY3-36, peptide devoid of Y1 receptor affinity. The Dark Agouti rat strain used in the present study has been shown to exhibit high plasma DP4 activity [9]. The antagonistic in vitro action of BIBO3304 additionally emphasized a role for Y1 receptor in NPY-induced enhancement of granulocyte adherence. However, the finding that Y2 (BIIE0246) and Y5 (L152804) receptor antagonists also abolished potentiating effect of NPY could be related to their suppressive effect on granulocyte adherence capacity.
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Extravasation of leukocytes into inflamed tissues is dependent on cells adherence to endothelium and the extracellular matrix. However, it is not clear whether increased adhesiveness slows down movement of leukocytes and delays emigration of cells or, alternatively, it is decrease in cell adhesiveness that actually reduces cells yield at the inflammatory site. It has been demonstrated that infusion of NPY in endotoxemic rats reduced tissue immigration of monocytes that involved Y2 receptor increase of adhesion characteristic of these cells [25]. Regardless of in vivo suppressive effect of NPY and NPY-related peptides on cells adherence capacity only NPY reduced their accumulation into the air pouch, suggesting involvement of an additional mechanism. The effect of NPY in vivo on inflammatory cells accumulation could be achieved through activation of Y receptors on blood vessels [36]. Taken together, anti-inflammatory action of NPY after in vivo application might be attained through the modulation of acute inflammation phases related to cells migration into inflamed tissue, and encompass both Y1 and Y2 receptors mediated effects. Phagocytosis of bacteria and foreign particles is a critical step for the successful elimination of pathogen. Concerning the in vitro effects of NPY, NPY-related peptides, and Y1, Y2 and Y5 receptor antagonists on granulocytes and monocytes phagocytic capacity, our results revealed Y1/Y5 receptor and Y2 receptor mediated potentiation and suppression of phagocytosis, respectively. Again, the striking decrease of phagocytic capacity of inflammatory cells following in vivo treatment with NPY could be a consequence of high plasma DP4 activity, which disallowed Y1 receptor-mediated potentiation and permitted Y2 receptor-mediated suppression of phagocytosis. However, we have shown before that NPY in vitro decreased phagocytic capacity of carageenan-elicited air pouch granulocytes via Y1 receptors in Albino Oxford rat strain [7]. Inflammatory and peripheral blood granulocytes could differ in many aspects including cell activation state and Y receptor expression. Besides, NPY in vitro decreased phagocytic capacity of human granulocytes via Y1/Y2 receptors [2]. Our earlier study discerned involvement of Y2 receptors in NPY-induced suppression of phagocytosis in rat peritoneal macrophages [10]. Our findings that: (i) NPY in the same way modulated adherence and phagocytic capacity of peripheral blood granulocytes and monocytes; and (ii) air-pouch cells consisted of roughly 80% of granulocytes (HIS48+CD11b+ cells) and 15% of monocytes (HIS48CD11b+ cells), advised that opposing effect of NPY on adherence and phagocytic capacity following in vitro and in vivo treatment was not a consequence of differences in the cells populations used in these studies. In conclusion, the modulation of rat inflammatory cells functions by NPY depends on the interaction between different Y receptor subtypes expressed on these cells. Specifically, the stimulatory effects of NPY are primarily exerted via Y1 receptor, while Y2/Y5 receptors mediate inhibitory effects of NPY. The differences between in vivo and in vitro effects of NPY on rat inflammatory cells functions are mostly due to dipeptidyl peptidase 4 activity. In addition, suppressive effect of NPY in vivo is highly dependent on the local microenvironment, peptide truncation and specific Y receptors interplay. Author contributions K.M., S.S. and M.D. designed the study and wrote the manuscript. S.S. supervised experiments. K.M., N.K. and V.V. were involved in experimental procedures performance. N.K. performed the analysis on FACScan flow cytometer. K.M. and M.D. performed statistical analysis of the data. All authors approved the final manuscript.
Conflicts of interest statement All authors state that they have no conflicts of interest regarding this study. Acknowledgement This work was supported by Grant (175050) from the Ministry of Science and Technological Development, Belgrade, Serbia. References [1] Ahmed AA, Wahbi AH, Nordlin K. Neuropeptides modulate a murine monocyte/macrophage cell line capacity for phagocytosis and killing of Leishmania major parasites. Immunopharmacol Immunotoxicol 2001;23:397–409. [2] Bedoui S, Kromer A, Gebhardt T, Jacobs R, Raber K, Dimitrijevic M, et al. Neuropeptide Y receptor-specifically modulates human neutrophil function. J Neuroimmunol 2008;195:88–95. [3] Bedoui S, Lechner S, Gebhardt T, Nave H, Beck-Sickinger AG, Straub RH, et al. NPY modulates epinephrine-induced leukocytosis via Y-1 and Y-5 receptor activation in vivo: sympathetic co-transmission during leukocyte mobilization. J Neuroimmunol 2002;132:25–33. [4] Bedoui S, Miyake S, Lin Y, Miyamoto K, Oki S, Kawamura N, et al. Neuropeptide Y (NPY) suppresses experimental autoimmune encephalomyelitis: NPY1 receptor-specific inhibition of autoreactive Th1 responses in vivo. J Immunol 2003;171:3451–8. [5] Bradley PP, Priebat DA, Christensen RD, Rothstein G. Measurement of cutaneous inflammation: estimation of neutrophil content with an enzyme marker. J Invest Dermatol 1982;78:206–9. [6] De la Fuente M, Bernaez I, Del Rio M, Hernanz A. Stimulation of murine peritoneal macrophage functions by neuropeptide Y and peptide YY. Involvement of protein kinase C. Immunology 1993;80:259–65. [7] Dimitrijevic M, Stanojevic S, Micic S, Vujic V, Kovacevic-Jovanovic V, Mitic K, et al. Neuropeptide Y (NPY) modulates oxidative burst and nitric oxide production in carrageenan-elicited granulocytes from rat air pouch. Peptides 2006;27:3208–15. [8] Dimitrijevic M, Stanojevic S, Mitic K, Kustrimovic N, Vujic V, Miletic T, et al. The anti-inflammatory effect of neuropeptide Y (NPY) in rats is dependent on dipeptidyl peptidase 4 (DP4) activity and age. Peptides 2008;29:2179–87. [9] Dimitrijevic M, Stanojevic S, Mitic K, Kustrimovic N, Vujic V, Miletic T, et al. Modulation of granulocyte functions by peptide YY in the rat: age-related differences in Y receptors expression and plasma dipeptidyl peptidase 4 activity. Regul Peptides 2010;159:100–9. [10] Dimitrijevic M, Stanojevic S, Vujic V, Beck-Sickinger A, von Horsten S. Neuropeptide Y and its receptor subtypes specifically modulate rat peritoneal macrophage functions in vitro: counter regulation through Y1 and Y2/5 receptors. Regul Peptides 2005;124:163–72. [11] El Karim IA, Lamey PJ, Linden GJ, Lundy FT. Neuropeptide Y Y1 receptor in human dental pulp cells of noncarious and carious teeth. Int Endod J 2008;41:850–5. [12] Felten DL, Felten SY, Carlson SL, Olschowka JA, Livnat S. Noradrenergic and peptidergic innervation of lymphoid tissue. J Immunol 1985;135:755s–65s. [13] Gehlert DR. Multiple receptors for the pancreatic polypeptide (PP-fold) family: physiological implications. Proc Soc Exp Biol Med 1998;218:7–22. [14] Groneberg DA, Folkerts G, Peiser C, Chung KF, Fischer A. Neuropeptide Y (NPY). Pulm Pharmacol Ther 2004;17:173–80. [15] Hassani H, Lucas G, Rozell B, Ernfors P. Attenuation of acute experimental colitis by preventing NPY Y1 receptor signaling. Am J Physiol Gastrointest Liver Physiol 2005;288:G550–6. [16] Hernanz A, Tato E, De la Fuente M, de Miguel E, Arnalich F. Differential effects of gastrin-releasing peptide, neuropeptide Y, somatostatin and vasoactive intestinal peptide on interleukin-1 beta, interleukin-6 and tumor necrosis factor-alpha production by whole blood cells from healthy young and old subjects. J Neuroimmunol 1996;71:25–30. [17] Ilhan A, Rasul S, Dimitrov A, Handisurya A, Gartner W, Baumgartner-Parzer S, et al. Plasma neuropeptide Y levels differ in distinct diabetic conditions. Neuropeptides 2010;44:485–9. [18] Kamath AB, Nagarkatti PS, Nagarkatti M. Characterization of phenotypic alterations induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin on thymocytes in vivo and its effect on apoptosis. Toxicol Appl Pharmacol 1998;150:117–24. [19] Levite M, Cahalon L, Hershkoviz R, Steinman L, Lider O. Neuropeptides, via specific receptors, regulate T cell adhesion to fibronectin. J Immunol 1998;160:993–1000. [20] Lin Q, Zou X, Ren Y, Wang J, Fang L, Willis WD. Involvement of peripheral neuropeptide Y receptors in sympathetic modulation of acute cutaneous flare induced by intradermal capsaicin. Neuroscience 2004;123:337–47. [21] Liu J, Li J, Zhai N, Geng L, Song F. Detection of the levels of neuropeptides, ACTH and cortisol in the blood of patients with polymyositis/dermatomyositis and their significance. J Dermatol 2004;31:392–7. [22] Medina S, Del Rio M, Manuel Victor V, Hernanz A, De la Fuente M. Changes with ageing in the modulation of murine lymphocyte chemotaxis by CCK-8S, GRP and NPY. Mech Ageing Dev 1998;102:249–61.
K. Miti´c et al. / Peptides 32 (2011) 1626–1633 [23] Mentlein R. Dipeptidyl-peptidase IV (CD26)—role in the inactivation of regulatory peptides. Regul Peptides 1999;85:9–24. [24] Michel MC, Beck-Sickinger A, Cox H, Doods HN, Herzog H, Larhammar D, et al. XVI. International Union of Pharmacology recommendations for the nomenclature of neuropeptide Y, peptide YY, and pancreatic polypeptide receptors. Pharmacol Rev 1998;50:143–50. [25] Nave H, Bedoui S, Moenter F, Steffens J, Felies M, Gebhardt T, et al. Reduced tissue immigration of monocytes by neuropeptide Y during endotoxemia is associated with Y2 receptor activation. J Neuroimmunol 2004;155: 1–12. [26] Pick E, Charon J, Mizel D. A rapid densitometric microassay for nitroblue tetrazolium reduction and application of the microassay to macrophages. J Reticuloendothel Soc 1981;30:581–93. [27] Reichlin S. Neuroendocrine–immune interactions. N Engl J Med 1993;329:1246–53. [28] Salomon J, Baran E. The role of selected neuropeptides in pathogenesis of atopic dermatitis. J Eur Acad Dermatol Venereol 2008;22:223–8. [29] Schaffer M, Beiter T, Becker HD, Hunt TK. Neuropeptides: mediators of inflammation and tissue repair? Arch Surg 1998;133:1107–16.
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[30] Stevens-Felten SY, Bellinger DL. Noradrenergic and peptidergic innervation of lymphoid organs. Chem Immunol 1997;69:99–131. [31] Straub RH, Mayer M, Kreutz M, Leeb S, Scholmerich J, Falk W. Neurotransmitters of the sympathetic nerve terminal are powerful chemoattractants for monocytes. J Leukoc Biol 2000;67:553–8. [32] Van Amersfoort ES, Van Berkel TJ, Kuiper J. Receptors, mediators, and mechanisms involved in bacterial sepsis and septic shock. Clin Microbiol Rev 2003;16:379–414. [33] von Horsten S, Ballof J, Helfritz F, Nave H, Meyer D, Schmidt RE, et al. Modulation of innate immune functions by intracerebroventricularly applied neuropeptide Y: dose and time dependent effects. Life Sci 1998;63:909–22. [34] Wheway J, Herzog H, Mackay F. NPY and receptors in immune and inflammatory diseases. Curr Top Med Chem 2007;7:1743–52. [35] Zhou JR, Xu Z, Jiang CL. Neuropeptide Y promotes TGF-beta1 production in RAW264.7 cells by activating PI3K pathway via Y1 receptor. Neurosci Bull 2008;24:155–9. [36] Zukowska Z, Pons J, Lee EW, Li L. Neuropeptide Y: a new mediator linking sympathetic nerves, blood vessels and immune system? Can J Physiol Pharmacol 2003;81:89–94.
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Peptides journal homepage: www.elsevier.com/locate/peptides
Neuropeptide Y modulates functions of inflammatory cells in the rat: Distinct role for Y1, Y2 and Y5 receptors Katarina Mitic´ a , Stanislava Stanojevic´ a , Nataˇsa Kuˇstrimovic´ a , Vesna Vujic´ b , Mirjana Dimitrijevic´ a,∗ a b
Institute of Virology, Vaccines and Sera, “Torlak”, Immunology Research Center “Branislav Jankovi´c”, Vojvode Stepe 458, 11152 Belgrade, Serbia Institute of Chemistry, School of Medicine, University of Belgrade, Belgrade, Serbia
a r t i c l e
i n f o
Article history: Received 21 April 2011 Received in revised form 7 June 2011 Accepted 7 June 2011 Available online 14 June 2011 Keywords: Granulocytes Inflammation Monocytes Neuropeptide Y Rat Y receptor subtypes
a b s t r a c t Neuropeptide Y (NPY) has been reported to be a potent anti-inflammatory peptide with ability to directly modulate activity of granulocytes and macrophages. The present study aimed to correlate the effects of NPY in vivo on lipopolysaccharide-induced air-pouch exudates cells and in vitro on peripheral blood leukocytes functions. The role of different Y receptors was examined using NPY-related peptides and antagonists with diverse subtype specificity and selectivity for Y receptors. Y1, Y2 and Y5 receptors were detected on air-pouch exudates cells (flow cytometry) and peripheral blood granulocytes (immunocitochemistry). NPY in vivo reduced inflammatory cells accumulation into the air pouch, and decreased their adherence and phagocytic capacity via Y2/Y5 and Y1/Y2 receptors, respectively. Quite the opposite, NPY in vitro potentiated adhesiveness and phagocytosis of peripheral blood granulocytes and monocytes by activating Y1 receptor. The differences between in vivo and in vitro effects of NPY on rat inflammatory cells functions are mostly due to dipeptidyl peptidase 4 activity. In addition, suppressive effect of NPY in vivo is highly dependent on the local microenvironment, peptide truncation and specific Y receptors interplay. © 2011 Elsevier Inc. All rights reserved.
1. Introduction Neuroendocrine derived factors in compliance with mediators originated from leukocytes and macrophages participate in the development and resolution of acute inflammation. Neuropeptides are responsible for the transmission of pain signals, regulation of vascular tonus and permeability, accumulation of inflammatory cells and stimulation of secretion by immune and endocrine cells [27,29]. The finding that inflammatory cells are located in the vicinity of nerve endings containing neuropeptides implicated that neuropeptides directly affect the functions of these cells [30]. Neuropeptide Y (NPY) is found in the sympathetic nerve endings and released upon sympathetic nervous system activation along with norepinephrine [12]. Over the last decade, an increasing number of evidences point to the involvement of NPY in the modulation of inflammatory reaction [8,11,15,20,34]. It has been shown that NPY in vitro enhanced
Abbreviations: NPY, neuropeptide Y; LP-NPY, [Leu31,Pro34]-NPY; hAAib-NPY, [hPP1-17, Ala31, Aib32]-NPY; LPS, lipopolysaccharide; DP4, dipeptidyl peptidase 4; NBT, nitro blue tetrazolium chloride; PBS, phosphate buffered saline; MEM, minimal essential medium without phenol red; MPO, myeloperoxidase; ODs, optical densities. ∗ Corresponding author. Tel.: +381 11 3976 674; fax: +381 11 2471 838. ´ E-mail address: [email protected] (M. Dimitrijevic). 0196-9781/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2011.06.007
functions of inflammatory cells such as adherence [6,25] chemotaxis [6,31], cytokine secretion [16,35] and production of reactive oxygen metabolites [2,6,10] and nitric oxide [7,8]. Some inhibitory effects of NPY on inflammatory cells activity, like reduction of granulocyte oxidative burst [7] and phagocytosis [2,7] have also been reported. NPY exerts biological activity via all subtypes of Y receptors, which are the Y1-5 receptor subtypes, while the y6 receptor has not been identified in rats [13]. An unusual feature of Y receptor family is the lack of sequence identity, particularly between the Y1, Y2 and Y5 receptor subtypes, which share only 30% sequence homology between themselves [24]. In addition to tissue-specific receptor subtype expression, NPY actions may be regulated by its processing by dipeptidyl peptidase 4 (DP4, also known as CD26). DP4 enzyme cleaves NPY1-36 to an Y2/5 receptor preferring peptide NPY3-36, therefore terminating its function on Y1 receptor [23]. A fine regulation of inflammatory cell activity by NPY is achieved through the engagement of different Y receptor subtypes mediating the opposing effects on a specific function, or simply by reducing the effect achieved through the activation of another receptor subtype [7,10]. Furthermore, NPY and/or Y receptors have been related to a number of inflammatory and immune mediated disorders [4,14,15,17,21,28]. Considering many conditions in which levels of NPY might be increased, it is of a great importance to discern its influence on immune and inflammatory cells residing in different compartments. The present study investigated the effect of NPY
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Fig. 1. Characterization of air-pouch exudates cells by flow cytometry: (a) percentage of cells expressing HIS48 and CD11b antigens; (b) representative dot plot showing the expression of HIS48 and CD11b antigens; (c) percentage of cells expressing Y1, Y2 and Y5 receptors; (d) mean fluorescence intensity (MFI) for Y1, Y2 and Y5 receptors; and (e) representative histogram plots of Y1, Y2 and Y5 receptors expression (black line) with negative control samples (omitted primary antibody, gray line). Statistically significant difference (ANOVA): *p < 0.05, MFI for Y5 receptor vs. Y1 and Y2 receptor.
Fig. 2. Immunocytochemical staining of rat peripheral blood granulocytes using anti-Y1-receptor, anti-Y2-receptor, anti-Y5-receptor and anti-CD11b molecule antibodies: (a) Y1-receptor positive cells (brown); (b) Y2-receptor positive cells (brown); (c) Y5-receptor positive cells (brown); (d) CD11b molecule positive cells (light red). Original magnification – ×40. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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in vivo on lipopolysaccharide (LPS)-induced air-pouch exudates cells recruitment and their adherence and phagocytic capacity. LPS has been shown to induce rapid neutrophils infiltration at the inflammatory site [32] resulting in neutralization and/or elimination of Gram negative bacteria. Receptor specificity was examined using NPY-related peptides with different pharmacological specificity for Y receptor subtypes. Also, we have tested the effects of NPY and NPY-related peptides in vitro on activity of peripheral blood monocytes and granulocytes. The involvement of different Y receptor subtypes in NPY-induced modulation of monocyte and granulocyte activity has been further estimated following combined treatment of cells with NPY and selective antagonists of the Y1, Y2 and Y5 receptors.
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2.3. Air-pouch induction and cell harvesting Air-pouches were created by three subcutaneous injections of sterile air into the dorsal region over the period of 7 days. On day 8, rats received one injection into the air pouch containing 10 g of peptide (NPY, Y1,2,4,5 receptor agonist; LP-NPY, Y1,5 receptor agonist; NPY3-36, Y2,5 receptor agonist; NPY13-36, Y2 receptor agonist; or hAAib-NPY, Y5 receptor agonist) and 20 g LPS in 2 ml of PBS. Control rats were injected with 20 g LPS in 2 ml of PBS. Cells were obtained by air-pouch lavage with 10 ml of PBS, 3 h later, washed three times in PBS, and adjusted to 2.5 × 106 or 1 × 107 cells/ml with regard to the assay. 2.4. Isolation of monocytes and granulocytes from peripheral blood Rats were bled by cardiac puncture in isoflurane anesthesia and euthanized immediately after. Briefly, heparinized blood was mixed with Dextran T500 (6% w/v in PBS) in order to obtain leukocyte enriched plasma. Mononuclear cells and granulocytes were separated by density gradient centrifugation over Percoll
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Human/rat NPY (Y1,2,4,5 receptor agonist), [Leu31,Pro34]-NPY (LP-NPY, Y1,5 receptor agonist), NPY3-36 (Y2,5 receptor agonist) and NPY13-36 (Y2 receptor agonist) were obtained from Polypeptide Laboratories, Wolfenbuttel, Germany. The Y5 receptor agonist [hPP1-17, Ala31 , Aib32 ]-NPY (hAAib-NPY) was a gift from Prof. Annette G. Beck-Sickinger, University of Leipzig, Germany. BIBO3304 (Y1 receptor antagonist), and BIIE0246 (Y2 receptor antagonist) were kindly provided by Dr. Henry Doods, Boehringer Ingelheim, Biberach, Germany. L152804, Y5 receptor antagonist, was obtained from Tocris (Bristol, UK). Lipopolysaccharide (LPS from E. coli) and zymosan were purchased from Sigma (St. Louis, MO, USA). Nitro blue tetrazolium chloride (NBT) was obtained from Serva (Heidelberg, Germany). Phosphate buffered saline (PBS) and Minimal essential medium without phenol red (MEM) were obtained from Institute “Torlak”, Belgrade, Serbia.
*
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OD (492 nm) x 1000
Female Dark Agouti rats, 4 months of age were obtained from the breeding colony at Immunology Research Center “Branislav ´ Belgrade (Serbia). The animals were housed individuJankovic”, ally in standard macrolone cages with free access to food pellets and tap water. All procedures involving the animals and their care were approved by our Institutional Animal Care and Use Committee and followed principles described in the European Community’s Council Directive of 24 November 1986 (86/609/EEC).
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Fig. 3. The effects of direct injection of NPY, LP-NPY, NPY3-36, NPY13-36 and hAAibNPY (10 g/20 g LPS/pouch) into the air pouch on: (a) total cell recruitment; (b) adherence capacity of cells stimulated with 1 g/ml of LPS; and (c) phagocytic capacity of cells stimulated with 125 g/ml of zymosan. The number of rats per group was eight. Statistically significant differences (t-test): *p < 0.05, and # p < 0.001, vs. control.
( = 1.077 g/ml). Granulocytes were isolated from pellets after lysis of erythrocytes with isotonic solution of NH4 Cl. 2.5. Flow cytometry For immunophenotyping, 1 × 106 air-pouch exudates cells per sample were washed and resuspended in flow cytometry buffer (PBS supplemented with 0.09% NaN3 ). All subsequent incubations were performed at +4 ◦ C in the dark, 30 min in duration, and were followed by through washings. Double staining of cells was performed using the fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-conjugated antibodies. The cells were directly stained with previously titrated mouse monoclonal anti granulocyte (HIS48) IgM-PE, anti-rat CD11b IgG-FITC (clone ED8) and anti-rat CD26 IgG-PE (clone Ox61). The cells were kept at +4 ◦ C in the dark prior to the analysis.
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Fig. 4. The in vitro effects of NPY, LP-NPY, NPY3-36 and hAAib-NPY at 10 M concentration on adherence capacity of peripheral blood granulocytes (a) and monocytes (b) stimulated with 1 g/ml of LPS. The number of cell samples per treatment was eight. Statistically significant differences (t-test): *p < 0.05, **p < 0.01, and # p < 0.001, vs. control.
For detection of Y receptors, cells were stained with polyclonal goat-anti Y receptor IgG (Y1 receptor, D-16; Y2 receptor, L-17; and Y5 receptor, N-20) followed by FITC-conjugated rabbit antigoat antibody. For the intracellular antigen immunolabeling, the cells were fixed with 0.25% paraformaldehide and permeabilized by 0.2% Tween 20 (15 min, +4 ◦ C) prior to the addition of antirat Y1 and Y2 antibodies, followed by FITC-conjugated anti-goat antibody as a secondary reagent. Antibodies were purchased from Becton Dickinson, San Jose CA, USA (CD11b-FITC, CD26-PE), Santa Cruz Biotechnology, Santa Cruz CA, USA (HIS48-PE and Y1, Y2 and Y5 receptors), and Sigma–Aldrich Chemie, Taufkirchen, Germany (rabbit anti-goat IgG-FITC). After extensive washing in flow cytometry buffer, all samples were analyzed on a FACScan flow cytometer using CellQuest software (Becton Dickinson, Mountain View, CA, USA). In addition, Y1, Y2 and Y5 receptor expression was analyzed by estimation of the mean fluorescence intensity (MFI), which correlates, in a relative sense, to the number of molecules expressed by the cell [18].
N-20) and mouse anti-rat CD11b IgG. The LSAB+ system was used to detect anti-rat Y receptor antibodies binding, while the EnVision G|2 Doublestain System was used to visualize antirat CD11b receptor antibody binding. After endogenous alkaline phosphatase and peroxidase activity was quenched, cells were washed in PBS and subsequently incubated with anti-rat primary antibodies diluted in background reducing antibody diluent (DakoCytomation) overnight at 4 ◦ C. Binding of the primary antibody was detected using appropriate secondary reagents and either 3,3 -diaminobenzidine (DAB+) (LSAB+ kit) or permanent red as chromogen (EnVision kit). Finally, cytospins were hematoxylin counterstained and cover-slipped. To verify staining specificity, the primary antibodies were substituted with antibody diluent. No specific immunoreactivity was detected in these cytospin preparations. A BH2 research microscope (Olympus Optical Co., Ltd., Tokyo, Japan) equipped with a ColorView III digital camera (Olympus) and Analysis Docu software (Olympus) was used to acquire images.
2.6. Immunocytochemistry
2.7. Adherence assay
Aliquots (105 cells) of peritoneal exudates cells were cytospun onto slides, fixed in ice-cold acetone and then frozen at −20 ◦ C. Prior to immunostaining, they were thawed, washed in phosphate buffered saline (PBS) and when necessary permeabilized with 0.5% saponin in PBS. For visualization of antibody binding LSAB+ and EnVison G|2 Doublestain (DakoCytomation, Glostrup, Denmark) kits were used according to the manufacturer’s instructions. The cells were stained with polyclonal goat-anti Y receptor IgG (Y1 receptor, D-16; Y2 receptor, L-17; and Y5 receptor,
The assay for adherence capacity was based on the measurement of myeloperoxidase (MPO) activity of cells adhered to tissue culture plates [5]. Briefly, individual air-pouch cell suspensions were adjusted to 2.5 × 106 cells/ml and plated at 50 l/well in 96well flat-bottomed tissue culture plates (NUNC, Roskilde, Denmark) previously filled with 50 l/well of 2 g/ml of LPS in MEM. Plates were incubated 60 min at 37 ◦ C in 95% air–5% CO2 , and nonadherent cells were removed by washing the wells twice with MEM. MPO activity was determined using ortho-phenylen-diamine in cit-
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Fig. 5. The effects of BIBO3304 (antagonist of Y1 receptor, 10−6 M), BIIE0246 (antagonist of Y2 receptor, 10−6 M) and L152804 (antagonist of Y5 receptor, 10−6 M) on NPY (10−6 M)-induced increase of adherence capacity of peripheral blood granulocytes (a) and monocytes (b) stimulated with 1 g/ml LPS. The number of cell samples per group was eight. Statistically significant differences (one-factor ANOVA for repeated measures followed by Fisher’s PLSD test; factor: treatment): *p < 0.05, **p < 0.01, # p < 0.001, treatment with specific antagonist (BIBO3304, BIIE0246 or L152804) or NPY vs. none; and a p < 0.05, treatment with NPY and specific antagonist (BIBO3304, BIIE0246 or L152804) vs. treatment with NPY.
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Fig. 6. The in vitro effects of NPY, LP-NPY, NPY3-36 and hAAib-NPY at 10 M concentration on phagocytic capacity of peripheral blood granulocytes (a) and monocytes (b) stimulated with 125 g/ml of zymosan. The number of cell samples per treatment was eight. Statistically significant differences (t-test): *p < 0.05, and **p < 0.01, vs. control.
ric buffer activated with 12% H2 O2 and Triton X-100. The reaction was stopped with 2 M H2 SO4 solution. Optical densities (ODs) were determined on Multiscan Ascent (Labsystems) at 492/620 nm. The results are expressed as OD (492/620 nm) × 1000. Adherence capacity of peripheral blood granulocytes and monocytes was determined in the same manner, except that tissue culture plates were previously filled with 50 l/well of: peptides (NPY, LP-NPY, NPY3-36, hAAib-NPY); specific antagonists of Y receptors (BIBO3304, BIIE0246, L152804); or NPY combined with specific antagonists of Y receptors (NPY/BIBO3304, NPY/BIIE0246, NPY/L152804) in 2 g/ml of LPS in MEM. Drugs were added at final concentration of 1 × 10−6 M. 2.8. Phagocytosis assay The phagocytic capacity was indirectly determined by measuring oxidative burst commenced after engulfment of foreign particles. Specifically, the assay was based on the reduction of yellow NBT to blue formazan by superoxide anion released following phagocytosis of zymozan [26]. Therefore, the magnitude of NBT reduction was proportional to the amount of zymosan particles phagocyted by cells. Individual air-pouch cells suspensions were adjusted to 2.5 × 106 cell/ml, and plated at 100 l/well in 96-well flat-bottomed tissue culture plates. Following incubation for 2 h at 37 ◦ C in 95% air–5% CO2 nonadherent cells were removed by washing the wells twice with MEM. Adherent cells were stimulated with 125 g/ml of zymosan in the presence of 0.5 mg/ml of NBT for 30 min at 37 ◦ C in 95% air–5% CO2 . Cells were fixed with methanol, and ODs were determined on Multiscan Ascent (Labsystems) at 545 nm. The results are expressed as OD (545 nm) × 1000.
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2.9. Statistical analysis Data were analyzed by one-way ANOVA, followed by Fisher’s PLSD test and Student’s t-test. Differences are regarded as statistically significant if p < 0.05. Results are presented as mean ± SE. The statistics were performed using statistical package SPSS10.0 for Windows. 3. Results 3.1. Characterization of air-pouch exudates cells Since the leukocyte integrin CD11b/CD18 (Mac-1; CR3) participates in different phases of inflammation, including the adherence and phagocytosis, the expression of both CD11b and granulocyte marker HIS48 on air-pouch exudates cells were evaluated (Fig. 1). Double staining of cells with HIS48 and CD11b antibodies confirmed that air-pouch exudates cells population elicited with LPS generally consisted of granulocytes (HIS48+CD11b+; 81.0 ± 1.1%) and monocytes (HIS48-CD11b+; 16.2 ± 2.1%), whereas approxi-
(b) *
*
100
a
* a
80 60 40 20
OD (545 nm ) x 1000
OD (545 nm ) x 1000
(a) 140
Phagocytic capacity of peripheral blood granulocytes and monocytes was determined in the same manner, except that adherent cells were treated with 100 l/well of: peptides (NPY, LP-NPY, NPY3-36, hAAib-NPY); specific antagonists of Y receptors (BIBO3304, BIIE0246, L152804); or NPY combined with specific antagonists of Y receptors (NPY/BIBO3304, NPY/BIIE0246, NPY/L152804) in 125 g/ml of zymozan. Peptides were added at concentration of 10−10 M, and antagonists of Y receptors at 100 time higher concentration, i.e. 10−8 M.
350 300
NPY**
a
NPY+
# a
250 200 150 100 50
0
0 none
BIBO3304 BIIE0246 L152804 −8
none
BIBO3304 BIIE0246 L152804 −8
Fig. 7. The effects of BIBO3304 (antagonist of Y1 receptor, 10 M), BIIE0246 (antagonist of Y2 receptor, 10 M) and L152804 (antagonist of Y5 receptor, 10−8 M) on NPY (10−10 M)-induced increase of phagocytic capacity of peripheral blood granulocytes (a) and monocytes (b) stimulated with 125 g/ml of zymosan. The number of cell samples per group was eight. Statistically significant differences (one-factor ANOVA for repeated measures followed by Fisher’s PLSD test; factor: treatment): *p < 0.05, **p < 0.01, # p < 0.001, treatment with specific antagonist (BIBO3304, BIIE0246 or L152804) or NPY vs. none; and a p < 0.05, treatment with NPY and specific antagonist (BIBO3304, BIIE0246 or L152804) vs. treatment with NPY.
K. Miti´c et al. / Peptides 32 (2011) 1626–1633
mately 3% of HIS48-CD11b-cells represented lymphocytes (Fig. 1a). Representative dot plot of double staining of cells with HIS48-PE and CD11b-FITC is shown in Fig. 1b. Given that activated T lymphocytes express membrane CD26, i.e. DP4 enzyme which is involved in the regulation of NPY biological activity, the presence of CD26+ cells among air-pouch exudates cells was tested by single staining with CD26 antibody. Analysis revealed that less than 3% of air-pouch exudates cells were CD26+ (data not shown). Single staining with antibodies specific for different Y receptor subtypes revealed that Y1, Y2 and Y5 receptors were expressed on roughly 5–8% of air-pouch exudates cells, Fig. 1c. Although there were no differences in the percentage of cells expressing different Y receptor subtypes, mean fluorescence intensity (MFI) for Y5 receptor was significantly lower in comparison with MFI for Y1 and Y2 receptors (Fig. 1d). Representative dot plots of double staining of cells with HIS48-PE and antibodies specific for different Y receptor subtypes are given in Fig. 1e. Immunocytochemical staining of rat peripheral blood granulocytes showed cells expressing Y1, Y2 and Y5 receptors as well as CD11b molecule (Fig. 2). As Y1 and Y2 antibodies recognized intracellular receptor domain in the vicinity of C-terminus, Fig. 2a and b illustrated staining of the inner cell membrane with Y1 and Y2 receptor antibody, respectively. In addition, Fig. 3b represented membrane staining with Y5 receptor antibody that was raised against an extracellular domain of the Y5 receptor. Membrane staining of cells with CD11b antibody was shown in Fig. 2d.
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accompanied with suppressive effect of antagonist per se on cells adherence capacity.
3.4. The effect of NPY and NPY-related peptides in vitro on phagocytic capacity of peripheral blood granulocytes and monocytes NPY (Y1,2,4,5 receptor agonist) and LP-NPY (Y1,5 receptor agonist) at concentration of 10−10 M significantly increased phagocytosis of zymosan in both granulocytes (Fig. 6a) and monocytes (Fig. 6b) from peripheral blood. In contrast, NPY3-36 (Y2,5 receptor agonist) reduced phagocytic capacity of monocytes. Y5 receptor agonist, hAAib-NPY, did not modulate phagocytosis of zymosan, neither in monocytes nor in granulocytes. These findings suggested the role for Y1 receptors in NPY-induced potentiation of phagocytosis in peripheral blood cells. However, both BIBO3304 (Y1 receptor antagonist, 10−8 M) and L152804 (Y5 receptor antagonist, 10−8 M) antagonized NPY-induced increase of phagocytic capacity of granulocytes (Fig. 7a) and monocytes (Fig. 7b). Also, BIBO3304 and L152804 itself, had suppressive effects on zymosan phagocytosis in peripheral blood granulocytes (Fig. 7a). On the other hand, BIBO3304 did not affect, whereas L152804 increased phagocytic capacity of monocytes (Fig. 7b).
4. Discussion 3.2. The effect of NPY and NPY-related peptides in vivo on inflammatory response to LPS NPY (Y1,2,4,5 receptor agonist, 10 g), directly injected into the air-pouch notably decreased LPS-induced cell recruitment, while the same dose of different NPY-related peptides had no effect on the migration of inflammatory cells (Fig. 3a). Finding that LP-NPY (Y1,5 receptor agonist) and hAAib-NPY (Y5 receptor agonist) slightly decrease and increase cells accumulation, respectively, suggested opposite function for Y1 and Y5 receptors regarding cells recruitment into the air pouch. Additionally, ineffectiveness of NPY3-36 (Y2,5 receptor agonist) and NPY13-36 (Y2 receptor agonist) in the modulation of inflammatory cells recruitment discarded the contribution of Y2 receptors. However, NPY and all NPY-related peptides used in the study decreased adherence capacity of inflammatory cells (Fig. 3b). The most pronounced inhibitory effect on adherence capacity of inflammatory cells from air pouch was observed following treatment with NPY3-36 (Y2,5 receptor agonist). Besides, suppressive effect on phagocytic function of air-pouch cells was detected after treatment with NPY and NPY-related peptides with exception for Y5 receptor agonist hAAib-NPY (Fig. 3c). 3.3. The effect of NPY and NPY-related peptides in vitro on adherence capacity of peripheral blood granulocytes and monocytes NPY, at concentration of 10−6 M, in vitro potentiated adherence capacity of LPS-stimulated peripheral blood granulocytes (Fig. 4a), and monocytes (Fig. 4b). Conversely, NPY3-36 (Y2,5 receptor agonist) and hAAib-NPY (Y5 receptor agonist) markedly reduced adherence capacity of granulocytes (Fig. 4a) whereas LP-NPY (Y1,5 receptor agonist) and hAAib (Y5 receptor agonist) decreased adherence of monocytes (Fig. 4b). Furthermore, BIBO3304, BIE0246 and L152804, specific antagonists of Y1, Y2 and Y5 receptors, respectively, antagonized NPY-induced increase of granulocyte adherence capacity (Fig. 5a). NPY-induced increase of monocyte adherence capacity was antagonized by BIE0246 and L152804 (Fig. 5b). It should be noticed that antagonistic action of pharmacological concentrations of BIBO3304, BIE0246 and L152804 (10−6 M) was
Reduced accumulation of inflammatory cells along with their decreased adherence and phagocytic capacity, caused by the in vivo administration of NPY into the inflamed tissue, sustained previously reported anti-inflammatory action of NPY [7–9,33]. Since air-pouch exudates cells mainly consisted of granulocytes, and small but significant proportion of granulocytes expressed Y1, Y2 and Y5 receptors, there was a possibility that NPY affected cells migration through Y receptor-specific mechanism. In view of different Y receptor specificity of LP-NPY (Y1,5 agonist), NPY3-36 (Y2,5 agonist), NPY13-36 (Y2 agonist) and hAAib-NPY (Y5 agonist) the finding that none of them affected cells infiltration in the air pouch advised that NPY suppressed cells accumulation by concomitant activation of Y1 and Y2 receptors. Y1 receptors on peripheral blood mononuclear cells mediated the inhibition of leukocyte mobilization following intravenous NPY in the rat [3]. Nevertheless, earlier studies showed that the effect of NPY on cells migration was dependant on the cell type. Particularly, NPY in vitro enhanced migration of leukocytes [6,22] and suppressed it in monocyte/macrophage cell line Raw 264.7 [1], but itself served as a chemoattractant for human monocytes [31]. In the present study NPY in vitro increased cell adherence via Y1 receptor but peptides devoid of Y1 receptor activity decreased it. Results viewed as a whole suggested that Y1 and Y2/Y5 receptors mediated potentiation and suppression of granulocyte adherence capacity, respectively. In contrast, it has been reported that NPYinduced rise in adherence capacity of rat monocytes [25] and human T lymphocytes [19] was mediated via Y2 receptors. The absence of Y1 receptor-mediated increase of inflammatory cells adhesion following in vivo treatment with NPY may have originated from DP4-induced conversion of NPY to NPY3-36, peptide devoid of Y1 receptor affinity. The Dark Agouti rat strain used in the present study has been shown to exhibit high plasma DP4 activity [9]. The antagonistic in vitro action of BIBO3304 additionally emphasized a role for Y1 receptor in NPY-induced enhancement of granulocyte adherence. However, the finding that Y2 (BIIE0246) and Y5 (L152804) receptor antagonists also abolished potentiating effect of NPY could be related to their suppressive effect on granulocyte adherence capacity.
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Extravasation of leukocytes into inflamed tissues is dependent on cells adherence to endothelium and the extracellular matrix. However, it is not clear whether increased adhesiveness slows down movement of leukocytes and delays emigration of cells or, alternatively, it is decrease in cell adhesiveness that actually reduces cells yield at the inflammatory site. It has been demonstrated that infusion of NPY in endotoxemic rats reduced tissue immigration of monocytes that involved Y2 receptor increase of adhesion characteristic of these cells [25]. Regardless of in vivo suppressive effect of NPY and NPY-related peptides on cells adherence capacity only NPY reduced their accumulation into the air pouch, suggesting involvement of an additional mechanism. The effect of NPY in vivo on inflammatory cells accumulation could be achieved through activation of Y receptors on blood vessels [36]. Taken together, anti-inflammatory action of NPY after in vivo application might be attained through the modulation of acute inflammation phases related to cells migration into inflamed tissue, and encompass both Y1 and Y2 receptors mediated effects. Phagocytosis of bacteria and foreign particles is a critical step for the successful elimination of pathogen. Concerning the in vitro effects of NPY, NPY-related peptides, and Y1, Y2 and Y5 receptor antagonists on granulocytes and monocytes phagocytic capacity, our results revealed Y1/Y5 receptor and Y2 receptor mediated potentiation and suppression of phagocytosis, respectively. Again, the striking decrease of phagocytic capacity of inflammatory cells following in vivo treatment with NPY could be a consequence of high plasma DP4 activity, which disallowed Y1 receptor-mediated potentiation and permitted Y2 receptor-mediated suppression of phagocytosis. However, we have shown before that NPY in vitro decreased phagocytic capacity of carageenan-elicited air pouch granulocytes via Y1 receptors in Albino Oxford rat strain [7]. Inflammatory and peripheral blood granulocytes could differ in many aspects including cell activation state and Y receptor expression. Besides, NPY in vitro decreased phagocytic capacity of human granulocytes via Y1/Y2 receptors [2]. Our earlier study discerned involvement of Y2 receptors in NPY-induced suppression of phagocytosis in rat peritoneal macrophages [10]. Our findings that: (i) NPY in the same way modulated adherence and phagocytic capacity of peripheral blood granulocytes and monocytes; and (ii) air-pouch cells consisted of roughly 80% of granulocytes (HIS48+CD11b+ cells) and 15% of monocytes (HIS48CD11b+ cells), advised that opposing effect of NPY on adherence and phagocytic capacity following in vitro and in vivo treatment was not a consequence of differences in the cells populations used in these studies. In conclusion, the modulation of rat inflammatory cells functions by NPY depends on the interaction between different Y receptor subtypes expressed on these cells. Specifically, the stimulatory effects of NPY are primarily exerted via Y1 receptor, while Y2/Y5 receptors mediate inhibitory effects of NPY. The differences between in vivo and in vitro effects of NPY on rat inflammatory cells functions are mostly due to dipeptidyl peptidase 4 activity. In addition, suppressive effect of NPY in vivo is highly dependent on the local microenvironment, peptide truncation and specific Y receptors interplay. Author contributions K.M., S.S. and M.D. designed the study and wrote the manuscript. S.S. supervised experiments. K.M., N.K. and V.V. were involved in experimental procedures performance. N.K. performed the analysis on FACScan flow cytometer. K.M. and M.D. performed statistical analysis of the data. All authors approved the final manuscript.
Conflicts of interest statement All authors state that they have no conflicts of interest regarding this study. Acknowledgement This work was supported by Grant (175050) from the Ministry of Science and Technological Development, Belgrade, Serbia. References [1] Ahmed AA, Wahbi AH, Nordlin K. Neuropeptides modulate a murine monocyte/macrophage cell line capacity for phagocytosis and killing of Leishmania major parasites. Immunopharmacol Immunotoxicol 2001;23:397–409. [2] Bedoui S, Kromer A, Gebhardt T, Jacobs R, Raber K, Dimitrijevic M, et al. Neuropeptide Y receptor-specifically modulates human neutrophil function. J Neuroimmunol 2008;195:88–95. [3] Bedoui S, Lechner S, Gebhardt T, Nave H, Beck-Sickinger AG, Straub RH, et al. NPY modulates epinephrine-induced leukocytosis via Y-1 and Y-5 receptor activation in vivo: sympathetic co-transmission during leukocyte mobilization. J Neuroimmunol 2002;132:25–33. [4] Bedoui S, Miyake S, Lin Y, Miyamoto K, Oki S, Kawamura N, et al. Neuropeptide Y (NPY) suppresses experimental autoimmune encephalomyelitis: NPY1 receptor-specific inhibition of autoreactive Th1 responses in vivo. J Immunol 2003;171:3451–8. [5] Bradley PP, Priebat DA, Christensen RD, Rothstein G. Measurement of cutaneous inflammation: estimation of neutrophil content with an enzyme marker. J Invest Dermatol 1982;78:206–9. [6] De la Fuente M, Bernaez I, Del Rio M, Hernanz A. Stimulation of murine peritoneal macrophage functions by neuropeptide Y and peptide YY. Involvement of protein kinase C. Immunology 1993;80:259–65. [7] Dimitrijevic M, Stanojevic S, Micic S, Vujic V, Kovacevic-Jovanovic V, Mitic K, et al. Neuropeptide Y (NPY) modulates oxidative burst and nitric oxide production in carrageenan-elicited granulocytes from rat air pouch. Peptides 2006;27:3208–15. [8] Dimitrijevic M, Stanojevic S, Mitic K, Kustrimovic N, Vujic V, Miletic T, et al. The anti-inflammatory effect of neuropeptide Y (NPY) in rats is dependent on dipeptidyl peptidase 4 (DP4) activity and age. Peptides 2008;29:2179–87. [9] Dimitrijevic M, Stanojevic S, Mitic K, Kustrimovic N, Vujic V, Miletic T, et al. Modulation of granulocyte functions by peptide YY in the rat: age-related differences in Y receptors expression and plasma dipeptidyl peptidase 4 activity. Regul Peptides 2010;159:100–9. [10] Dimitrijevic M, Stanojevic S, Vujic V, Beck-Sickinger A, von Horsten S. Neuropeptide Y and its receptor subtypes specifically modulate rat peritoneal macrophage functions in vitro: counter regulation through Y1 and Y2/5 receptors. Regul Peptides 2005;124:163–72. [11] El Karim IA, Lamey PJ, Linden GJ, Lundy FT. Neuropeptide Y Y1 receptor in human dental pulp cells of noncarious and carious teeth. Int Endod J 2008;41:850–5. [12] Felten DL, Felten SY, Carlson SL, Olschowka JA, Livnat S. Noradrenergic and peptidergic innervation of lymphoid tissue. J Immunol 1985;135:755s–65s. [13] Gehlert DR. Multiple receptors for the pancreatic polypeptide (PP-fold) family: physiological implications. Proc Soc Exp Biol Med 1998;218:7–22. [14] Groneberg DA, Folkerts G, Peiser C, Chung KF, Fischer A. Neuropeptide Y (NPY). Pulm Pharmacol Ther 2004;17:173–80. [15] Hassani H, Lucas G, Rozell B, Ernfors P. Attenuation of acute experimental colitis by preventing NPY Y1 receptor signaling. Am J Physiol Gastrointest Liver Physiol 2005;288:G550–6. [16] Hernanz A, Tato E, De la Fuente M, de Miguel E, Arnalich F. Differential effects of gastrin-releasing peptide, neuropeptide Y, somatostatin and vasoactive intestinal peptide on interleukin-1 beta, interleukin-6 and tumor necrosis factor-alpha production by whole blood cells from healthy young and old subjects. J Neuroimmunol 1996;71:25–30. [17] Ilhan A, Rasul S, Dimitrov A, Handisurya A, Gartner W, Baumgartner-Parzer S, et al. Plasma neuropeptide Y levels differ in distinct diabetic conditions. Neuropeptides 2010;44:485–9. [18] Kamath AB, Nagarkatti PS, Nagarkatti M. Characterization of phenotypic alterations induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin on thymocytes in vivo and its effect on apoptosis. Toxicol Appl Pharmacol 1998;150:117–24. [19] Levite M, Cahalon L, Hershkoviz R, Steinman L, Lider O. Neuropeptides, via specific receptors, regulate T cell adhesion to fibronectin. J Immunol 1998;160:993–1000. [20] Lin Q, Zou X, Ren Y, Wang J, Fang L, Willis WD. Involvement of peripheral neuropeptide Y receptors in sympathetic modulation of acute cutaneous flare induced by intradermal capsaicin. Neuroscience 2004;123:337–47. [21] Liu J, Li J, Zhai N, Geng L, Song F. Detection of the levels of neuropeptides, ACTH and cortisol in the blood of patients with polymyositis/dermatomyositis and their significance. J Dermatol 2004;31:392–7. [22] Medina S, Del Rio M, Manuel Victor V, Hernanz A, De la Fuente M. Changes with ageing in the modulation of murine lymphocyte chemotaxis by CCK-8S, GRP and NPY. Mech Ageing Dev 1998;102:249–61.
K. Miti´c et al. / Peptides 32 (2011) 1626–1633 [23] Mentlein R. Dipeptidyl-peptidase IV (CD26)—role in the inactivation of regulatory peptides. Regul Peptides 1999;85:9–24. [24] Michel MC, Beck-Sickinger A, Cox H, Doods HN, Herzog H, Larhammar D, et al. XVI. International Union of Pharmacology recommendations for the nomenclature of neuropeptide Y, peptide YY, and pancreatic polypeptide receptors. Pharmacol Rev 1998;50:143–50. [25] Nave H, Bedoui S, Moenter F, Steffens J, Felies M, Gebhardt T, et al. Reduced tissue immigration of monocytes by neuropeptide Y during endotoxemia is associated with Y2 receptor activation. J Neuroimmunol 2004;155: 1–12. [26] Pick E, Charon J, Mizel D. A rapid densitometric microassay for nitroblue tetrazolium reduction and application of the microassay to macrophages. J Reticuloendothel Soc 1981;30:581–93. [27] Reichlin S. Neuroendocrine–immune interactions. N Engl J Med 1993;329:1246–53. [28] Salomon J, Baran E. The role of selected neuropeptides in pathogenesis of atopic dermatitis. J Eur Acad Dermatol Venereol 2008;22:223–8. [29] Schaffer M, Beiter T, Becker HD, Hunt TK. Neuropeptides: mediators of inflammation and tissue repair? Arch Surg 1998;133:1107–16.
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[30] Stevens-Felten SY, Bellinger DL. Noradrenergic and peptidergic innervation of lymphoid organs. Chem Immunol 1997;69:99–131. [31] Straub RH, Mayer M, Kreutz M, Leeb S, Scholmerich J, Falk W. Neurotransmitters of the sympathetic nerve terminal are powerful chemoattractants for monocytes. J Leukoc Biol 2000;67:553–8. [32] Van Amersfoort ES, Van Berkel TJ, Kuiper J. Receptors, mediators, and mechanisms involved in bacterial sepsis and septic shock. Clin Microbiol Rev 2003;16:379–414. [33] von Horsten S, Ballof J, Helfritz F, Nave H, Meyer D, Schmidt RE, et al. Modulation of innate immune functions by intracerebroventricularly applied neuropeptide Y: dose and time dependent effects. Life Sci 1998;63:909–22. [34] Wheway J, Herzog H, Mackay F. NPY and receptors in immune and inflammatory diseases. Curr Top Med Chem 2007;7:1743–52. [35] Zhou JR, Xu Z, Jiang CL. Neuropeptide Y promotes TGF-beta1 production in RAW264.7 cells by activating PI3K pathway via Y1 receptor. Neurosci Bull 2008;24:155–9. [36] Zukowska Z, Pons J, Lee EW, Li L. Neuropeptide Y: a new mediator linking sympathetic nerves, blood vessels and immune system? Can J Physiol Pharmacol 2003;81:89–94.