Expression of peptide YY by human blood leukocytes

Peptides 58 (2014) 78–82

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Expression of peptide YY by human blood leukocytes Julia Pia Natascha Holler a,∗ , Jessica Schmitz a,1 , Rainer Roehrig b,2 , Sigrid Wilker a,1 , Andreas Hecker a,1 , Winfried Padberg a,1 , Veronika Grau a,1 a

Laboratory of Experimental Surgery, Department of General and Thoracic Surgery, Justus-Liebig-University, Feulgen-Str. 10-12, D-35385 Giessen, Germany Department of Anesthesiology, Intensive Care and Pain Medicine, Justus-Liebig-University of Giessen and Marburg, Campus Giessen, Rudolf-Bucheim-Straße 7, 35392 Giessen, Germany b

a r t i c l e

i n f o

Article history: Received 18 March 2014 Received in revised form 19 May 2014 Accepted 20 May 2014 Available online 23 June 2014 Keywords: Neuropeptide Y Pancreatic polypeptide Inflammation Differentiation

a b s t r a c t Peptide YY is produced by L cells in the mucosa of the distal ileum, colon, and rectum and may have systemic and paracrine functions. We hypothesized that peptide YY is expressed by peripheral blood mononuclear cells. The purpose of the present study was to evaluate the expression of peptide YY mRNA and peptide by peripheral blood mononuclear cells and differentiated THP-1 cells after lipopolysaccharide treatment as an in vitro model of inflammation. Blood was drawn by venipuncture from 18- to 63-yearold healthy male blood donors (n = 63); peptide YY mRNA expression levels were detected in peripheral blood mononuclear cells from all healthy male subjects. In 3 subjects, peripheral blood mononuclear cells were cultured for 3 and 24 h and peptide YY was detected in the cell culture supernatant. In human monocytic THP-1 cells treated with phorbol-12-myristate-13-acetate to induce differentiation to macrophages, treatment with lipopolysaccharide caused down-regulation of peptide YY mRNA levels. In summary, freshly isolated peripheral blood mononuclear cells from healthy humans expressed peptide YY. In vitro data suggested that peptide YY expression is down-regulated by differentiation of monocytes to macrophages and proinflammatory stimuli. © 2014 Elsevier Inc. All rights reserved.

1. Introduction Neuropeptide Y, peptide YY (PYY), and pancreatic polypeptide are members of the neuropeptide Y family of peptides that evolved by gene duplication, and they share structural homology [2,20,25]. These peptides are major regulators of the gut–brain axis that regulates digestion, uptake of nutrients, and appetite [20]. Neuropeptide Y is expressed as a sympathetic cotransmitter of neuronal cells in the central nervous system and periphery, and PYY and pancreatic polypeptide are produced by endocrine cells of the gut and pancreas [20]. Full-length neuropeptide Y and PYY bind to receptors Y1, Y2, Y4, and Y5, and pancreatic polypeptide binds to receptor Y4 [7,18]. Therefore, peptides of the neuropeptide Y family may have

∗ Corresponding author. Tel.: +49 641 985 44701; fax: +49 641 985 44709. E-mail addresses: [email protected] (J.P.N. Holler), [email protected] (J. Schmitz), [email protected] (R. Roehrig), [email protected] (S. Wilker), [email protected] (A. Hecker), [email protected] (W. Padberg), [email protected] (V. Grau). 1 Tel.: +49 641 985 44701; fax: +49 641 985 44709. 2 Tel.: +49 641 985 44401; fax: +49 641 985 44409. http://dx.doi.org/10.1016/j.peptides.2014.05.009 0196-9781/© 2014 Elsevier Inc. All rights reserved.

overlapping cellular functions. Neuropeptide Y and PYY are cleaved by dipeptidyl peptidase 4 to form truncated neuropeptide Y3–36 and PYY3–36 that have reduced affinity for the Y1 receptor and increased affinity for the Y2 receptor [28]. The peptide PYY is produced by L cells in the mucosa of the distal ileum, colon, and rectum. These cells release PYY into the bloodstream and the gastrointestinal tract lumen when they are exposed to unabsorbed nutrients [8]. Therefore, PYY may have systemic and paracrine functions. Release of PYY reduces gastrointestinal motility, decreases gastric acid secretion, and increases nutrient absorption via neuropeptide Y receptors [6]. Furthermore, administration of PYY may protect animals from developing hapten-induced colitis (US Patent application US 2006/0122106A1). In addition, in an animal model of induced pancreatitis, exogenous PYY may suppress inflammation by decreasing cytokine and amylase release [37,38]. Analogs of PYY have been developed that are potent inhibitors of pancreatic cancer proliferation in vitro and in vivo, and PYY and its analogs may inhibit the growth of breast, esophageal, and gastric cancer in vitro [1]. The 2 major forms of PYY are PYY1–36 and PYY3–36 . The most common form of circulating PYY is PYY3–36 , which binds with high affinity to the Y2 receptor. In animals, PYY3–36 has an anorectic effect that is mediated by the Y2 receptor. In rats, PYY is produced by a discrete population of neurons localized in the medulla

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oblongata and islets of Langerhans [17]. Neuropeptide Y acts as a neurotransmitter and regulates various physiologic functions including appetite, mood, and vasoconstriction [16]. The functions of neuropeptide Y in the modulation of inflammatory and immunomodulatory reactions have become more reasonable. Furthermore, neuropeptide Y increases functions of immune cells in vitro such as adhesion, chemotaxis, cytokine secretion, and production of reactive oxygen metabolites and nitric oxide [29]. Neuropeptide Y also may inhibit some inflammatory cell activities and may decrease granulocyte oxidative burst and phagocytosis. In animal studies, neuropeptide Y reduces inflammatory cell accumulation, adhesion, and phagocytic capacity that are mediated by Y2/Y5 and Y1/Y2 receptors. Beyond the brain–gut axis, numerous additional functions have been attributed to neuropeptide Y including regulation of innate and adaptive immunity [14]. Leukocytes may express receptors Y1, Y2, Y4, and Y5 and neuropeptide Y may regulate cell migration, antigen presentation, T-cell polarization, and diverse effector functions [14]. We showed that peripheral blood mononuclear cells (PBMC) of the rat express high levels of neuropeptide Y mRNA and peptide, similar to sympathetic ganglia [19], suggesting that neuropeptide Y may function as an autocrine or paracrine regulator of leukocytes. Leukocytic neuropeptide Y may be sensed by neurons. Marked down-regulation of neuropeptide Y expression by mononuclear leukocytes was observed in vivo, and these leukocytes may accumulate in the blood vessels of renal allografts undergoing acute rejection, which is a vigorous Th1/M1-type immune reaction [19]. In contrast with rat leukocytes, human steady-state blood leukocytes are not an abundant source of neuropeptide Y [15,33]. Upon stimulation of human leukocytes with T-cell mitogens or nerve growth factor in vitro, neuropeptide Y mRNA expression is induced [10,26,33]. Alterations in fluid and tissue concentrations of neuropeptide Y and PYY may occur, suggesting that these peptides may have similar functions. However, a relation between these alterations has not been identified previously [21,32,34]. In human PBMC, neuropeptide Y expression is very low (unpublished data). However, PYY and neuropeptide Y may transmit signals via common receptors. Therefore, we hypothesized that PYY is expressed by PBMC. The purpose of the present study was to evaluate the expression of PYY mRNA and peptide by PBMC and differentiated THP-1 cells after lipopolysaccharide (LPS) treatment as an in vitro model of inflammation. 2. Material and methods 2.1. Healthy human blood donors After approval by the ethics committee of the University of Giessen (approval No. 67/11) and informed consent, blood was drawn by veni puncture from 18- to 63-year-old healthy male blood donors (n = 63) (Table 1). Data about age, body weight, body height, body mass index, allergies, smoking, alcohol consumption, medication, blood pressure, body temperature, and medical history were

Table 1 Characteristics of healthy male subjects.a Characteristic

Median (range) or number (%)

Age (years) Body mass index Smoking Moderate alcohol consumption Allergies

42 (18–63) 26 (23–49) 27 (43) 8 (13) 12 (19)

a N = 63 subjects. Data reported as mean (range, minimum to maximum) or number (%).

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collected. Exclusion criteria were acute or chronic disease, cancer history, medications, alcohol consumption (3 times/week), recent surgical procedure, blood pressure > 130/90 mm Hg, body temperature (measured in the ear) > 37 ◦ C, or previous organ transplant. Volunteers were defined as smokers when they smoked daily. Alcohol consumption ≤3 times/week was defined as moderate. The PBMC were isolated by gradient centrifugation (Ficoll-Paque Plus, Amersham Biosciences Europe, Freiburg, Germany) for 10 min at 1000 × g at room temperature [9]. 2.2. THP-1 cells The THP-1 cells (DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany) were adjusted to 2.5 × 105 cells/mL and cultivated in Roswell Park Memorial Institute 1640 medium (Pasching, Austria) at 37 ◦ C and 5% carbon dioxide in a humidified incubator. Differentiation to macrophages was induced by addition of phorbol-12-myristate13-acetate (PMA) (Sigma–Aldrich, Saint Louis, MO, USA) at a final concentration of 100 ng/mL. At 3 days after addition of PMA, the medium was replaced by fresh Roswell Park Memorial Institute 1640 containing 1 ␮g/mL LPS (Sigma–Aldrich) and cells were collected after 3, 6, 12, 24, and 48 h. Cells were lysed (RNA Lysis Buffer T, Peqlab, Erlangen, Germany), snap frozen, and stored at −80 ◦ C until RNA isolation for real-time polymerase chain reaction (RTPCR). 2.3. RNA isolation and polymerase chain reaction Total RNA was isolated from PBMC from human male healthy subjects and THP-1 cells (RNeasy Mini Kit, Qiagen, Hilden, Germany). RNA was reverse transcribed using the M-MLV H− reverse transcriptase and random hexamer primers (Promega, Mannheim, Germany). Real-time PCR was performed in duplicates (ABI 7700 Sequence Detection System, Applied Biosystems, Foster City, CA, USA; Platinum SYBR green qPCR Super Mix-UDG, Invitrogen, Karlsruhe, Germany). Primer pairs for human pseudogene-free porphobilinogen deaminase, human neuropeptide Y (A and B), human PYY, and human pancreatic polypeptide were used (Table 2). Primers were selected for intron spanning cDNA sequences, synthesized commercially (MWG Biotech, Ebersberg, Germany), and used at a concentration of 0.6 ␮M. Nested RT-PCR was performed to detect neuropeptide Y mRNA. The PCR included initial denaturation for 5 min at 95 ◦ C, 45 cycles of 20 s at 60 ◦ C, and 10 s at 72 ◦ C. In each experiment, negative controls were included (cDNA replaced by water). No product was obtained in negative controls. Human pancreatic tissue was used as positive control. To evaluate the PCR products, the melting curves were analyzed and products were separated in agarose gels, stained with ethidium bromide, and sequenced (MWG Biotech) to confirm the identity of the amplicon. Expression data for PBMC were reported as CT values. The data from THP-1 cells were calculated as 2−CT , where CT was the difference in CT values between the gene of interest and porphobilinogen deaminase. Data for each experimental group were expressed in relation to the expression by control treated for 3 h with PMA the absence of LPS, which was set to 1 arbitrary unit (AU). 2.4. Measurement of peptide YY To prove that the PBMC expressed PYY mRNA and secreted PYY peptide, PBMC were purified by gradient centrifugation from different healthy male volunteers (n = 3; 1 nonsmoker and 2 smokers; age, 42, 48, and 56 years; regular body mass index; moderate alcohol consumption). The PBMC were incubated at 6 × 106

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Fig. 1. Gel electrophoresis of mRNA amplified by real-time polymerase chain reaction (PCR). Data are shown for pancreatic polypeptide (PPY), peptide YY (PYY), and neuropeptide Y (NPY) mRNA expression by human peripheral blood mononuclear cells of healthy male subjects (data from 3 representative subjects shown). The PCR product was separated in agarose gels to confirm product size and homogeneity. M, marker (100 bp DNA ladder ranging from 100 bp to 1 kbp); P, human pancreatic tissue; C, negative control with cDNA replaced by water.

Table 2 Primers used for real-time polymerase chain reaction. Gene

Primer

Sequence

Amplicon size (bp)

Neuropeptide Y A (NC 000007.13)

Sense antisense

206

Neuropeptide Y B (NC 000007.13)

Sense antisense

Porphobilinogen deaminase (NC 000011.9)

Sense antisense

Pancreatic polypeptide Y (NC 000017.11)

Sense antisense

Peptide YY (NM 001034080.1)

Sense antisense

5 -ACGCACCAGCGGAGGACAT-3 5 -AGGCCAGAGAGCAAGTCTCAT-3 5 -CGCTGCGACACTACATCAAC-3 5 -GGTCTTCAAGCCGAGTTCTG-3 5 -CCCACGCGAATCACTCTCAT-3 5 -TGTCTGGTAACGGCAATGCG-3 5 -GCGTGGCTCTGTTACTACAG-3 5 -GTATCTACGGAGATCAGCTGC-3 5 -GGACACGCTTCTTTCCAAAACG-3 5 -TTCTGGGGTCGGGAGTGCGTATGC-3

162 70 122 158

cells/mL in Roswell Park Memorial Institute 1640 medium without supplements. Cell culture supernatant (500 ␮L) was removed after 3 and 24 h and PYY level measured by enzyme-linked immunosorbent assay (Millipore, Billerica, MA, USA). This assay measured human PYY3–36 (specificity, 100%) and PYY1–36 (specificity, 104%), but did not cross-react with neuropeptide Y or pancreatic polypeptide. 2.5. Statistical analyses Data analysis was performed with statistical software (IBM SPSS Statistics for Windows, Version 20.0, IBM Corp., Armonk, NY, USA). Exploratory analysis of human data was performed with Pearson product moment correlation with uncorrected P values. Results from THP-1 cells were reported as median and percentile (0, 25, 75, and 100), presented in box plots, and analyzed with Kruskal–Wallis test and Mann–Whitney test. Statistical significance was defined by P ≤ .05. 3. Results 3.1. Neuropeptide Y, peptide YY, and pancreatic polypeptide mRNA expression in peripheral blood mononuclear cells from healthy donors Neuropeptide Y and pancreatic polypeptide mRNA were detected only in a subpopulation of blood donors (neuropeptide Y, 42 donors [67%]; pancreatic polypeptide, 36 donors [57%]). In human pancreatic tissue, which was used as a positive control, both transcripts were readily detected (Fig. 1). In individuals expressing neuropeptide Y or pancreatic polypeptide, expression levels were close to the detection limit. In contrast, high PYY mRNA expression levels were detected in PBMC from all healthy male subjects (n = 63) and no DNA amplification was observed in negative controls (Fig. 1). Slightly higher PYY mRNA expression levels were detected in older (age, 47–60 years) than younger donors (age, 18–46 years) (Fig. 2). No correlation was observed between PYY mRNA expression levels and body weight, body height, body mass index, smoking, or moderate alcohol consumption.

Fig. 2. Pearson product moment correlation of age-dependent peptide YY (PYY) mRNA expression by peripheral blood mononuclear cells from healthy men (n = 63) (r = 0.439; P = .01). The mRNA expression was measured by real-time polymerase chain reaction. Porphobilinogen deaminase was used as reference gene. Data are expressed as delta-CT and regression line is shown.

3.2. Release of peptide YY from human peripheral blood mononuclear cells The PBMC were cultured for 3 and 24 h, and PYY was detected in the cell culture supernatant (n = 3). Average PYY level was 7 ± 2 pg/mL after 3 h and 9 ± 3 pg/mL after 24 h (Table 3). Table 3 Release of peptide YY from human peripheral blood mononuclear cells in culture.a Subject age (years)

PYY (pg/mL)3 h

PYY (pg/mL)24 h

42 48 56

6.37 6.89 7.65

8.62 8.91 9.24

Mean ± SD

7±2

9±3

a

N = 3 subjects. Peptide YY (PYY) measured by enzyme-linked immunosorbent assay.

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Fig. 3. Real-time polymerase chain reaction of peptide YY (PYY) mRNA expression by THP-1 cells. Cells were treated with phorbol-12-myristate-13-acetate (PMA) in the absence or presence of lipopolysaccharide (LPS) for 3, 6, 12, 24, and 48 h. Data are expressed as arbitrary units that were normalized to a mean 1 for differentiated THP-1 cells in the absence of LPS. The box plots show median and percentiles (0, 25, 75, and 100); *P ≤ .05; n = 4 for each experiment.

3.3. Expression of peptide YY mRNA by THP-1-cells Although undifferentiated cells (n = 4) of the human monocytic THP-1 cell line had high PYY mRNA expression levels, no effect of LPS application was observed in these cells (data not shown). When THP-1 cells were differentiated to macrophages after the addition of PMA, significantly lower PYY mRNA expression levels were detected in comparison with undifferentiated controls (differentiated, 1.18 AU; undifferentiated, 1.0 AU; P ≤ .05). After treatment of differentiated THP-1 cells with LPS (n = 4), a timedependent down-regulation of PYY mRNA levels was observed after 12 h (differentiated, 1.0 AU; undifferentiated, 0.65 AU; P ≤ .04), 24 h (differentiated, 0.92 AU; undifferentiated, 0.34 AU; P ≤ .03) and 48 h (differentiated, 0.94 AU; undifferentiated, 0.78 AU; P ≤ .05) (Fig. 3). At 24 h after treatment with LPS, the lowest PYY expression (3-fold) was reached; at 48 h after LPS treatment, the PYY expression levels were low but recovered to normal levels. 4. Discussion In this study, we showed that freshly isolated PBMC from healthy male subjects expressed PYY mRNA and peptide, but neuropeptide Y and pancreatic polypeptide were not consistently expressed. In our cohort, PYY mRNA expression levels increased with age, but the baseline expression level of PYY in PBMC is unknown because confounding factors may have been present. Estrogen may modulate human monocytes and macrophages [24]. Men experience a greater severity and prevalence of bacterial and viral infections than women, who may exhibit a more robust response to vaccination [22,23]. Estrogen may modulate CD16 expression on human macophages and affect the production of interleukin 1␤, interleukin 6, and tumor necrosis factor [24]. In the present study, only men were evaluated to avoid possible effects of female sex hormones on leukocyte PYY expression. The THP-1 cells are premonocytes that are committed to the monocytic cell lineage. They grow in suspension and do not adhere to the plastic surfaces of culture plates. Addition of PMA induced differentiation to macrophage-like cells that adhered to the dish bottom and had morphologic characteristics of macrophages. In these differentiated THP-1 cells, PYY mRNA expression was downregulated by the proinflammatory bacterial cell wall component LPS. The results of PYY expression by human PBMC are consistent with previous findings in steadystate rat PBMC that abundantly

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expressed neuropeptide Y and down-regulated expression during strong inflammation [19]. Both peptides were expressed by monocytes (data not shown). Another similarity between rat leukocyte neuropeptide Y and human PYY is that both peptides are down-regulated by proinflammatory stimuli and differentiation to macrophages [19]. However, the functions of PYY expressed by human PBMC are unknown. Neuropeptide Y and PYY may have similar functions because they bind to the same receptors and are substrates of the same enzyme, dipeptidyl peptidase 4 [20]. Evidence that supports a pivotal immunomodulatory role of neuropeptide Y in PBMC includes the effects of neuropeptide Y in mobilizing mononuclear leukocytes from the marginal pool of the bloodstream, inhibiting tissue infiltration by monocytes, regulating the phagocytic activity of monocytes and macrophages, enabling Tcell activation by antigen presenting cells, and inhibiting Th1-type immunity [3–5,31,39]. Although information is limited about the immunomodulatory functions of PYY, anti-inflammatory effects of exogenous PYY have been shown in experimental pancreatitis and colitis (US Patent application US 2006/0122106A1) [38]. This is consistent with our findings in LPS-treated THP-1 cells, which is a model of inflammation, in which PYY expression was decreased. The mechanisms of these anti-inflammatory properties of PYY are unknown because the effects at the molecular level including receptor binding and specificity have been only partially resolved. However, we know that PYY binds to Y1 and Y2 and may share anti-inflammatory functions with its homologue neuropeptide Y. The present data suggest that PYY expression by human PBMC increases with age. Age-dependent changes have been described in rats including the expression of the neuropeptide Y family of peptides, expression, immunological functions of these peptides, and dipeptidyl peptidase 4 levels [11–14,27,36]. The present results are consistent with the known general suppression of innate and adaptive immunity during aging [30,35]. However, the data about age-dependent PYY expression should be viewed with caution because of the limited sample size. 5. Conclusion We showed that PYY was expressed in freshly isolated PBMC from healthy men. In vitro data suggested that PYY expression is down-regulated by differentiation of monocytes to macrophages and proinflammatory stimuli. These findings are evidence that neuroimmunologic interactions may be mediated by members of the neuropeptide Y family of peptides and suggest that PYY may be a pleiotropic cytokine produced in a regulated way by PBMC. Acknowledgements The authors thank Prof. Dr. G. Bein (Giessen, Germany) who provided blood samples, Prof. Dr. W. Kummer (Giessen, Germany) for helpful suggestions, and Prof. Dr. V. Fendrich (Marburg, Germany) who provided human pancreatic tissue. References [1] Alosi JA, McFadden DW. Peptide YY mediates inhibition of tumor growth and inflammation. Methods Mol Biol 2009;512:377–94. [2] Ballantyne GH. Peptide YY(1–36) and peptide YY(3–36): Part I. Distribution, release and actions. Obes Surg 2006;16:651–8. [3] Nuhlmann S, Nave H, Drube J, Pabst R, von Hörsten S. Differential effects of neuropeptide Y (NPY) on leukocyte subsets in the blood: mobilization of B-1-like B-lymphocytes and activated monocytes. J Neuroimmunol 2001;117:125–32. [4] Bedoui S, Kawamura N, Straub RH, Pabst R, Yamamura T, von Höorsten S. Relevance of neuropeptide Y for the neuroimmune crosstalk. J Neuroimmunol 2003;134:1–11. [5] Kuhlmann S, Nave H, Drube J, Pabst R, von Höorsten S. Differential effects of neuropeptide Y (NPY) on leukocyte subsets in the blood: mobilization of B-1-like B-lymphocytes and activated monocytes. J Neuroimmunol 2001;117:125–32.

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[6] Bedoui S, von Hörsten S, Gebhardt T. A role for neuropeptide Y (NPY) in phagocytosis: implications for innate and adaptive immunity. Peptides 2007;28:373–6. [7] Bird AR, Croom Jr WJ, Fan YK, Black BL, McBride BW, Taylor IL. Peptide regulation of intestinal glucose absorption. J Anim Sci 1996;74:2523–40. [8] Blomqvist AG, Herzog H. Y-receptor subtypes – how many more. Trends Neurosci 1997;20:294–8. [9] Böttcher G, Ekblad E, Ekman R, Håkanson R, Sundler F. Peptide YY: a neuropeptide in the gut. Immunocytochemical and immunochemical evidence. Neuroscience 1993;55:281–90. [10] Böyum A. Isolation of mononuclear cells and granulocytes from human blood. Isolation of monuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g. Scand J Clin Lab Invest Suppl 1968;97:77–89. [11] Bracci-Laudiero L, Aloe L, Stenfors C, Tirassa P, Theodorsson E, Lundberg T. Nerve growth factor stimulates production of neuropeptide Y in human lymphocytes. Neuroreport 1996;7:485–8. [12] De la Fuente M, Del Río M, Medina S. Changes with aging in the modulation by neuropeptide Y of murine peritoneal macrophage functions. J Neuroimmunol 2001;116:156–67. [13] De la Fuente M, Del Río M, Víctor VM, Medina S, Neuropeptide Y. effects on murine natural killer activity: changes with ageing and cAMP involvement. Regul Pept 2001;101:73–9. [14] De la Fuente M, Medina S, Del Rio M, Ferrández MD, Hernanz A. Effect of aging on the modulation of macrophage functions by neuropeptides. Life Sci 2000;67:2125–35. [15] 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 Pept 2010;159:100–9. [16] Ericsson A, Hemsén A, Lundberg JM, Persson H. Detection of neuropeptide Y-like immunoreactivity and messenger RNA in rat platelets: the effects of vinblastine, reserpine, and dexamethasone on NPY expression in blood cells. Exp Cell Res 1991;192:604–11. [17] Gehlert DR. Multiple receptors for the pancreatic polypeptide (PP-fold) family: physiological implications. Proc Soc Exp Biol Med 1998;218:7–22. [18] Glavas MM, Grayson BE, Allen SE, Copp DR, Smith MS, Cowley MA, et al. Characterization of brainstem peptide YY (PYY) neurons. J Comp Neurol 2008;506:194–210. [19] Herzog H, Darby K, Ball H, Hort Y, Beck-Sickinger A, Shine J. Overlapping gene structure of the human neuropeptide Y receptor subtypes Y1 and Y5 suggests coordinate transcriptional regulation. Genomics 1997;41:315–9. [20] Holler J, Zakrzewicz A, Kaufmann A, Wilhelm J, Fuchs-Moll G, Dietrich H, et al. Neuropeptide Y is expressed by rat mononuclear blood leukocytes and strongly down-regulated during inflammation. J Immunol 2008;181:6906–12. [21] Holzer P, Reichmann F, Farzi A. Neuropeptide Y, peptide YY and pancreatic polypeptide in the gut–brain axis. Neuropeptides 2012;46:261–74.

[22] Kaye WH, Berrettini W, Gwirtsman H, George DT. Altered cerebrospinal fluid neuropeptide Y and peptide YY immunoreactivity in anorexia and bulimia nervosa. Arch Gen Psychiatry 1990;47:548–56. [23] Klein SL. The effects of hormones on sex differences in infection: from genes to behavior. Neurosci Biobehav Rev 2000;24:627–38. [24] Klein SL, Poland GA. Personalized vaccinology: one size and dose might not fit both sexes. Vaccine 2013;31:2599–600. [25] Kramer PR, Kramer SF, Guan G. 17 beta-estradiol regulates cytokine release through modulation of CD16 expression in monocytes and monocyte-derived macrophages. Arthritis Rheum 2004;50:1967–75. [26] Larhammar D. Evolution of neuropeptide Y, peptide YY and pancreatic polypeptide. Regul Pept 1996;62:1–11. [27] Levite M. Neuropeptides, by direct interaction with T cells, induce cytokine secretion and break the commitment to a distinct T helper phenotype. Proc Natl Acad Sci U S A 1998;95:12544–9. [28] Medina S, Del Río M, Hernanz A, De la Fuente M, The NPY. effects on murine leukocyte adherence and chemotaxis change with age. Adherent cell implication. Regul Pept 2000;95:35–45. [29] Mentlein R, Dahms P, Grandt D, Krüger R. Proteolytic processing of neuropeptide Y and peptide YY by dipeptidyl peptidase IV. Regul Pept 1993;49: 133–44. [30] Mitic´ K, Stanojevic´ S, Kuˇstrimovic´ N, Vujic´ V, Dimitrijevic´ M. J Clin 2013;123:958–65. [31] 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. [32] Riviere PJ, Rao RK, Pascaud X, Junien JL, Porreca F. Effects of neuropeptide Y, peptide YY and sigma ligands on ion transport in mouse jejunum. J Pharmacol Exp Ther 1993;264:1268–74. [33] Schwarz H, Villiger PM, von Kempis J, Lotz M. Neuropeptide Y is an inducible gene in the human immune system. J Neuroimmunol 1994;51:53–61. [34] Sheikh SP. Neuropeptide Y and peptide YY: major modulators of gastrointestinal blood flow and function. Am J Physiol 1991;261:G701–15. [35] Solana R, Tarazona R, Gayoso I, Lesur O, Dupuis G, Fulop T. Innate immunosenescence: effect of aging on cells and receptors of the innate immune system in humans. Semin Immunol 2012;24:331–41. ´ [36] Stanojevic´ S, Vujic´ V, Kovacevic-Jovanovi c´ V, Mitic´ K, Kosec D, Hörsten S, et al. Age-related effect of peptide YY (PYY) on paw edema in the rat: the function of Y1 receptors on inflammatory cells. Exp Gerontol 2006;41:793–9. [37] Tseng WW, Liu CD. Peptide YY and cancer: current findings and potential clinical applications. Peptides 2002;23:389–95. [38] Vona-Davis L, McFadden DW. PYY and the pancreas: inhibition of tumor growth and inflammation. Peptides 2007;28:334–8. [39] Wheway J, Mackay CR, Newton RA, Sainsbury A, Boey D, Herzog H, et al. A fundamental bimodal role for neuropeptide Y1 receptor in the immune system. J Exp Med 2005;202:1527–38.