Regulatory Peptides 159 (2010) 100–109

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Regulatory Peptides

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Modulation of granulocyte functions by peptide YY in the rat: Age-related differencesin Y receptors expression and plasma dipeptidyl peptidase 4 activity

Mirjana Dimitrijević a,⁎, Stanislava Stanojević a, Katarina Mitić a, Nataša Kuštrimović a, Vesna Vujić b,Tatjana Miletić a, Vesna Kovačević-Jovanović a

a Immunology Research Center “Branislav Janković”, Institute of Virology, Vaccines and Sera, “Torlak”, Vojvode Stepe 458, 11221 Belgrade, Serbiab Institute of Chemistry, School of Medicine, Belgrade, Serbia

⁎ Corresponding author. Tel.: +381 11 3976 674; faxE-mail address: miradim@sezampro.rs (M. Dimitrije

0167-0115/$ – see front matter © 2009 Elsevier B.V. Adoi:10.1016/j.regpep.2009.11.002

a b s t r a c t

a r t i c l e i n f o

Article history:Received 27 May 2009Received in revised form 9 October 2009Accepted 1 November 2009Available online 6 November 2009

Keywords:Dipeptidyl peptidase 4 (DP4)GranulocytesPeptide YY (PYY)Y receptorsRats

It has been acknowledged that aging exerts detrimental effects on cells of the innate immune system andthat neuropeptides, including neuropeptide Y (NPY) and NPY-related peptides fine-tune the activity of thesecells through a receptor specific mechanism. The present study investigated the age-dependent potential ofpeptide YY (PYY) to modulate different granulocyte functions. The PYY reduced the carrageenan-elicitedgranulocyte accumulation into the air-pouch of aged (24 months) rats, and markedly decreased thephagocytosis of zymosan, as well as the H2O2 production, when applied in vivo (20 μg/air-pouch). The anti-inflammatory effect of PYY was less prominent in adult (8 months) and young (3 months) rats. However, theproportions of granulocytes expressing Y1, Y2 and Y5 receptor subtypes were significantly lower in bothaged and young rats when compared to adult rats. Furthermore, the aging was found to be associated withthe diminished dipeptidyl peptidase 4 (DP4, an enzyme converting the NPY and PYY to Y2/Y5 receptorselective agonists) activity in plasma. In conclusion, the diverse age-related anti-inflammatory effect of PYYin rats originates from different expression levels of Y1, Y2, and Y5 receptor subtypes in addition to differentplasma DP4 activity.

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© 2009 Elsevier B.V. All rights reserved.

1. Introduction

Neuropeptide Y (NPY1–36), a widely present neuropeptide inmammals, is located in, and released from the specific central andperipheral non-adrenergic non-cholinergic (particularly enteric),sympathetic and sensory neurons [1]. In addition to its capacity toshift circadian rhythms, its proven hypertensive and potent appetite-stimulating effects, the NPY exhibits anxiolytic, anti-stress, anti-depressant, anti-convulsant and anti-nociceptive actions in thecentral nervous system [2–4]. Peptide YY (PYY1–36) is a member ofNPY family of peptides, primarily released from enteric endocrinecells, and involved in pancreatic secretion, gall bladder secretion andgut motility [5]. Even so, low amounts of PYY are found in certainsympathetic fibers, suggesting that it can exhibit neurotransmitterfunction as well. Apart from the distinction in their tissue/organdistribution, there is a difference in the affinity for NPY Y receptorsubtypes between the PYY and NPY. Specifically, the NPY is an agonistof all receptor subtypes, namely Y1, Y2, Y3, Y4, Y5 and Y6, while thePYY lacks the affinity for the proposed Y3 receptor, which has notbeen cloned yet [6–8]. However, it was demonstrated that PYY canmimic the effects of NPY in essentially all studies involving its

administration to experimental animals or the studies using thecloned receptors, or tissue preparations [3]. The biological activity ofboth PYY and NPY is controlled by their processing by dipeptidylpeptidase 4 (DP4, also known as CD26). DP4 cleaves NPY1–36 andPYY1–36 and produces the Y2/Y5 receptor preferring peptides, NPY3–36

and PYY3–36, but also terminates their function on Y1 receptor [9]. Anunusual feature of Y receptor family is their lack of sequence identity,particularly between the Y1, Y2 and Y5 receptor subtypes, whichshare only 30% sequence homology between themselves [8].

Numerous studies established the stimulatory effect of NPY on amultitude of macrophage functions such as adherence [10,11],chemotactic activity and phagocytosis [10], cytokine secretion [12],and reactive oxygen metabolites [10,12,13] and nitric oxide [14]production. Some inhibitory effects of the NPY on inflammatory cellsactivity, e.g. suppression of macrophage chemotactic response [15,16]and reduction of granulocyte oxidative burst and phagocytosis [17,18]have also been reported. Furthermore, the modulatory effects of NPYon immune and inflammatory cells functions appear to be age-dependent [12,19–22]. For instance, the NPY exhibited a stimulatoryeffect on monocyte-mediated phagocytosis in adult mice, while itshowed an inhibitory effect in aged mice [12].

Moreover, the pathophysiological role of NPY in infection andinflammation [23], as well as in autoimmunity has been suggested [24].An increased susceptibility to infections and inflammatory diseases dueto aging has been partly associated with alterations in the immune and

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inflammatory cells response to invading agents [25–27]. Although theage-related changes in the innate immune system have been less wellinvestigated compared to the adaptive immunity, the reduced phago-cytic capability and superoxide anion generation with aging have beenproved [28,29]. Additionally, changes in tissue microenvironment andthe neuroendocrinemilieumay also contribute to altered inflammatoryreaction in aged rodents and humans [30]. In that sense, changes inneuropeptide expression pattern and receptor density are regularlyobserved with aging, but also in various pathophysiological conditions.Recent findings by Donoso et al. suggested that a dysregulation in sym-pathetic neurotransmitter regulatorymechanismsmayplay a role in theimpairment of the inflammatory response during aging [31]. Specifi-cally, aged rats exhibited reduced serum levels of NPY and ATP, accom-panied with the markedly increased serum levels of TNFα and IL-10following the induction of acute inflammation. Furthermore, reducedexpression of both Y2 receptors and DP4 in spleens of aged mice hasbeen linked with impaired angiogenesis due to aging [32].

The present study aimed to determine the potential of PYY tomodulate granulocyte functions in experimental animals of differentage. For that purpose we have examined the effect of PYY, administeredin vivo and in vitro, on carrageenan-elicited air-pouch granulocyteadherence capacity, phagocytosis and H2O2 production in young(3 months), adult (8 months) and aged (24 months) rats. The phago-cytic capability was evaluated in cells stimulated with zymosanparticles. Following the binding of zymosan to integrin CD11b/CD18(also known as CR3, Mac-1), phagocytosis is initiated and consequentlyoxidative burst commences [33]. The level of H2O2 production wasdetermined in cells stimulated with phorbol myristate acetate (PMA)that directly activates protein kinase C (PKC) and triggers oxidativeburst without phagocytosis.

Since CD11b participates in different inflammation phases,including the adherence and phagocytosis, its expression on granu-locytes was also evaluated. Furthermore, granulocytes were examinedfor the presence of different NPY Y receptor subtypes, specifically Y1,Y2 and Y5 receptors. As the biological effects of PYY are regulated byenzyme DP4, the DP4-like activity in plasma, air-pouch exudates andcells, as well as the expression of CD26 molecule (exhibiting DP4-likeactivity) on air-pouch exudate cells, were tested in rats of differentage.

2. Methods

2.1. Animals

Male Albino Oxford (AO) rats, 3, 8 and 24 months of age (assignedas young, adult and aged, respectively) were obtained from thebreeding colony at Immunology Research Center “Branislav Janković”,Belgrade (Serbia). The animals were housed individually in standardmacrolone cages with free access to food pellets and tap water. Allprocedures involving the animals and their care were approved by ourInstitutional Animal Care and Use Committee and followed principlesdescribed in the European Community's Council Directive of 24November 1986 (86/609/EEC).

2.2. Drugs

Human/rat PYY (Y1,2,4,5 receptor agonist), [Leu31,Pro34]-NPY (LP-NPY, Y1,5 receptor agonist) and NPY3–36 (Y2,5 receptor agonist) wereobtained from Polypeptide Laboratories, Wolfenbuttel, Germany.BIBO 3304, Y1 receptor antagonist, and BIIE 0246, Y2 receptorantagonist, were kindly provided by Dr. Henry Doods, BoehringerIngelheim, Biberach, Germany. The reversible, competitive transitionstate analogue inhibitor of DP4, isoleucil thiazolidide (IleThia) waskindly provided by Dr. Hans-Ulrich Demuth (Probiodrug, Halle,Germany). Carrageenan, lipopolysaccharide (LPS from E. coli), phorbolmyristate acetate (PMA), zymosan (ZYM) and horseradish peroxidase

were purchased from Sigma (St. Louis, Mo., USA). Nitro bluetetrazolium chloride (NBT) was obtained from Serva (Heidelberg,Germany). Minimal essential mediumwithout phenol red (MEM)wasobtained from Institute “Torlak”, Belgrade, Serbia.

2.3. Air-pouch induction and cell harvesting

Air-pouches were created by three subcutaneous injections of sterileair into thedorsal regionover theperiodof7 days. Carrageenan, 20 mg/ratin 2 ml of phosphate buffered saline (PBS) was injected directly into theair pouch on day 8. For the in vivo treatment with PYY, rats received oneinjection containing20 μg of PYYand20 mgof carrageenan in 2 ml of PBS.Cells were obtained by air-pouch lavage with 2 ml of PBS, 24 h later. Air-pouch exudates containing cells were centrifuged (250 g, 10 min) andsupernatants were then used for determination of DP4 activity and H2O2

concentration. Cell pellets were resuspended and washed three times inPBS, and adjusted to 2.5×106 or 1×107 cells/ml.

2.4. Flow cytometry

For immunophenotyping, 1×106 air-pouch exudate cells per samplewere washed and resuspended in flow cytometry buffer (PBSsupplemented with 1 % bovine serum albumin, BSA, and 0.1 % NaN3).All subsequent incubations were performed at +4 °C in the dark,30 min in duration, and were followed by thorough washings. Doublestaining of cells was performed using the fluorescein isothiocyanate(FITC)- or phycoerythrin (PE)-conjugated antibodies. The cells weredirectly stained with previously titrated mouse monoclonal anti-granulocyte (HIS48) IgM-PE, anti-rat CD11b IgG-FITC (clone ED8), anti-rat CD26 IgG-PE (clone Ox61), and anti-rat CD3-Biotin (clone G4.18)followed by FITC-conjugated streptavidin (SAvFITC) as a second stepreagent. The cells were fixed in 0.5 ml of 1% paraformaldehyde and keptat 4 °C in the dark prior to the analysis. In a separate experiment cellswere double stained with HIS48 IgM-PE, followed by the intracellularstaining protocol using the anti-rat CD68-Biotin (clone ED1) andSAvFITC. The procedures were carried out as described for directstaining, except that for the intracellular antigen immunolabelling, thecells were fixed with 0.25% paraformaldehide and permeabilized by0.2% Tween 20 (15 min, ±4 °C) prior to addition of anti-rat ED1-Biotinantibody.

For detection of Y receptors, cells were double stained with HIS48IgM-PE and polyclonal goat-anti Y receptor IgG (Y1 receptor, D-16; Y2receptor, L-17; and Y5 receptor, N-20) followed by FITC-conjugatedrabbit anti-goat antibody. For the intracellular antigen immunolabel-ling, the cells were fixed and permeabilized prior to addition of anti-rat Y1 and Y2 antibodies, followed by FITC-conjugated anti-goat anti-body as a secondary reagent. Antibodies were purchased from BectonDickinson, San Jose CA, USA (CD11b-FITC, CD26-PE, CD3-Biotin,SAvFITC), Santa Cruz Biotechnology, Santa Cruz CA, USA (HIS48-PEand Y1, Y2 and Y5 receptors), Serotec, Oxford, UK (CD68-Biotin), andSigma-Aldrich Chemie, Taufkirchen, Germany (anti-goat IgG-FITC).After extensive washing in flow cytometry buffer, all samples wereanalyzed on a FACScan flow cytometer (Becton Dickinson, MountainView, CA, USA).

2.5. DP4-like enzymatic activity

Rats were bled in isoflurane anesthesia, and the blood sampleswere collected by cardiac puncture. Rats were euthanized immediate-ly after bleeding. EDTA plasma samples were kept at−80 °C until use.DP4-like enzymatic activity was determined at 37 °C by using thechromogenic substrate 0.5 mM Gly-Pro-p-nitroanilide in 50 mM Tris/HCl pH 8.0 in a 96-well microplate assay. The assay used 10 μl plasmasample, 80 μ l Tris–HCl and 10 μl substrate. The optical density (OD) ofreleased p-nitroaniline was measured at 405 nM at the beginning ofthe enzyme reaction (immediately following the substrate addition)

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and 60 min later. The DP4 activity in the samples was calculatedusing p-nitroaniline standard curve over the concentration range of1–80 μM. The results are expressed as mU/ml, where mU is defined asthe enzyme activity that releases one μM of p-nitroaniline from the

Fig. 1. The expression of HIS48 and CD11b antigens (A); HIS48 and ED1 (B); and CD26 and(24 months) rats. The representativedot plots showing theexpressionof CD11b (D) andED1(E)(F) and CD26+ED1+ (G) cells in air-pouch exudate cell suspensions. The number of rats per grand adult; cadult vs. young and aged.

substrate in 1 min. The same assay was used for determination ofDP4-like enzymatic activity in the air-pouch exudate, following theremoval of cells, and also in cells adhered on tissue culture plates asdescribed for the phagocytosis assay.

CD3 (C) on air-pouch exudate cells from young (3 months), adult (8 months) and agedonHIS48+air-pouch exudate cells, anddot plots illustrating thepresence of CD26+CD3+oupwas eight. Statistically significant differences, pb0.05: aaged vs. adult; baged vs. young

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2.6. Adherence

Tissue culture plates (96-well flat-bottomed, NUNC, Roskilde,Denmark) were filled with 50 μl/well of PYY (10−12 to 10−6 M), PYY(10−6 M)+BIBO 3304 (10−6 M) and BIBO 3304 (10−6 M) inMEMandcells 50 μl/well (2.5×106 cells/ml) were plated afterwards. Plates wereincubated (10, 20 and 60 min) at 37 °C in 95% air–5% CO2, and washedwith MEM. Cells were fixed with methanol, stained with 1% methyleneblue,washed throughoutwith distilledwater and dried overnight. Colorwas dissolved in 200 μl/well 0.1% HCl and OD were determined at620 nm [34]. Results are expressed as OD (620 nm)×1000.

2.7. Phagocytosis assay

Individual cell suspensions adjusted to 2.5×106 cell/ml wereplated at 100 μl/well in 96-well flat-bottomed tissue culture plates.Plates were incubated for 2 h at 37 °C in 95% air–5% CO2. Nonadherentcells were removed by washing the wells twice with MEM. Cellswere stimulated with ZYM (125 μg/ml) with or without the additionof PYY, LP-NPY or NPY3–36 (10−12 to 10−6 M) in the presence of NBT(0.5 mg/ml) for 30 min at 37 °C in 95% air–5% CO2 [35]. In anadditional experiment cells were incubated with PYY (10−8 M) withor without the addition of Ilethiazolidide, BIBO 3304 or BIIE 0246 at(10−6 M). Cells were fixed with methanol, and OD of blue formazanobtained after reduction of yellow NBT was determined at 545 nm.Results are expressed as OD (545 nm)×1000.

2.8. H2O2 assay

Individual cell suspensions were prepared in the same manner asdescribed for the phagocytosis assay. Granulocytes (adhered on 96-wellflat-bottomed tissue cultureplates)were stimulated forH2O2production

Fig. 2. The percentage of Y1, Y2 and Y5 receptor-positive granulocytes (HIS48+ cells) withinand aged (24 months) rats (A). The representative dot plots showing the expression of Y1 reof rats per group was eight. Statistically significant differences:apb0.001, adult vs. young an

with 25 nM PMA (1.54 ng/ml) in phenol red solution (140 mM NaCl,10 mM potassium phosphate buffer, pH 7, 5.5 mM dextrose, 0.56 mMphenol red and 19 U/ml horseradish peroxidase), with or without theaddition of PYY, LP-NPY or NPY3–36 (10−12 to 10−6 M) [36]. In anadditional experiment cells were incubatedwith PYY (10−10 M)with orwithout the addition of IleThia, BIBO 3304 or BIIE 0246 at (10−6 M). ThePMA working solution was prepared from 10−2 M PMA in DMSO stocksolution, stored at−70 °C. The plates were incubated for 1 h at 37 °C in95% air–5% CO2. The reaction was stopped by addition of 10 μl of 0.5 MNaOH per well, and the OD was determined at 620 nm. Theconcentration of H2O2 in the samples was calculated using a standardcurvewith the range of 1 to 40 μMof H2O2. The concentration of H2O2 inair-pouch exudate supernatants was determined after mixing 50 μl ofexudate supernatantwith 50 μl of phenol red solution (2×concentrated)and 10 μl of NaOH.

2.9. Statistical analysis

Data were analyzed by one-way ANOVA, followed by Fisher's PLSDtest and Student's t-test. Differences are regarded as statisticallysignificant if pb0.05. Results are presented asmean±SE. The statisticswere performed using statistical package SPSS10.0 for Windows.

3. Results

3.1. Characterization of air-pouch exudates cells

Since the leukocyte integrin Mac-1 (CD11b/CD18) participates indifferent inflammation phases, including the adherence and phago-cytosis, the expression of both CD11b and granulocyte marker HIS48on air-pouch exudate cells were evaluated. The analysis revealed thatthe majority of these cells expressed both granulocyte marker HIS48

the air-pouch exudate cell population derived from young (3 months), adult (8 months)ceptor on HIS48+ air-pouch exudate cells from adult (B) and aged (C) rats. The numberd aged.

Fig. 3. DP4-like activity in plasma (A) and air-pouch exudates (B) and adherent cells(C) obtained 24 h after challenge with carrageenan (20 mg) in young (3 months), adult(8 months) and aged (24 months) rats. The number of rats per group was eight.Statistically significant differences: apb0.001, young vs. adult and aged; bpb0.001, adultvs. aged; and cpb0.05, young vs. adult.

Fig. 4. The effect of direct injection of PYY (20 μg) into the air pouch on total cellrecruitment induced by carrageenan (20 mg) in young (3 months), adult (8 months)and aged (24 months) rats 24 h after challenge. The number of rats per groupwas eight.Statistically significant differences: apb0.05, control aged rats vs. control young andcontrol adult rats, and ⁎pb0.05, PYY vs. corresponding control.

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and CD11b marker (Fig. 1A). The proportion of HIS48+CD11b+ cellswas greater in aged rats when compared to adult rats (82% vs. 72%),while the percentage of HIS48−CD11b− was lower in aged rats inrelation to young and adult rats. Double staining of cells with HIS48and ED1 antibody confirmed that air-pouch exudate cell populationgenerally consisted of granulocytes, while a low percentage ofmacrophages was identified (Fig. 1B). The percentage of HIS48+granulocytes including both single (HIS48+ED1−) and double positive(HIS48+ED1+) cells was comparable in rats of different age (young,72.1±3.9%; adult, 76.3±1.8%; aged, 78.1±1.0%). Nevertheless, thesingle and double positive cell populations obtained from adult ratsdiffered to some extent from the cells obtained from both young andaged rats in terms of their different cell type proportion. For instance,lower percentage of macrophages (HIS48−ED1+) was detected inadult (4.9±0.4%) rats in comparison with young (7.7±0.6%) and aged(9.7±0.9%) rats. Moreover, the percentage of CD26+CD3− cells(representing CD26+non-T cells)was decreased in air-pouch exudatesfrom adult rats when compared to young and aged rats (Fig. 1C). Itshould be underlined that the percentage of T cells in air-pouchexudates was approximately 5%, less than 1% of total cells were positivefor both CD26 and CD3. The representative dot plots are shown in Fig. 1.D,E,F. However, the percentage of ED1−CD26+ cells (roughly 5%, asshown in dot plot, Fig. 1G) indicated that there were CD26+ cellspresent which did not belong to macrophage (ED1+), and most likelydid not belong to T cells population (less than 1% of CD3+CD26+cells).

3.2. Expression of Y1, Y2 and Y5 receptors on granulocytes

The percentage of granulocytes expressing Y1 receptors was sig-nificantly higher in adult rats in comparison with young and agedrats, while there were no differences between young and aged rats(Fig. 2A). Similar age-associated trend of receptor expression wasobserved for Y2 and Y5 receptor subtypes. Representative dot plots ofY1 receptor expression on granulocytes from adult (B) and aged (C)rats are incorporated in Fig. 2.

3.3. DP4-like activity

Plasma DP4-like activity significantly decreased with aging(Fig. 3A), the same being the case for DP4-like activity in the air-pouch exudates (Fig. 3B). A diminutive DP4-like activity detected inadherent cells recovered from air-pouch also declined with age(Fig. 3C).

3.4. The effect of PYY on cells recruitment and adherence

The inflammatory response to carrageenan observed in aged ratswas more intense compared to adult and young rats, as the number ofcells recovered from the air-pouch significantly increased in advancedage (Fig. 4). Furthermore, PYY (20 μg) injected into the air-pouchsignificantly reduced carrageenan-induced cell recruitment in agedrats only.

In adult rats, stimulationwith PYY in vitro increased the adherencecapacity of granulocytes after 20 min and 60 min of incubation only atthe highest concentration (10−6 M) used (Fig. 5A). The Y1 receptorantagonist BIBO 3304 (10−6 M) blocked the PYY-induced increase ofgranulocyte adherence from adult rats after 20 min (Fig. 5B), thusconfirming the role for Y1 receptor in the modulation of granulocyteadherence by PYY. Although granulocytes from young and adult ratsexpressed different adherence dynamics, their adherence capacitywas comparable when tested after 60 min (Fig. 5C). Adherencecapacity of granulocytes from aged rats was lower in comparison withadherence capacity of granulocytes from adult and aged rats; themoststriking difference was noted at 60 min. Furthermore, the in vitrotreatment with PYY (10−6 M) increased the adherence capacity ofgranulocytes from adult rats after 20 min and 60 min of incubation,

but had no effect on the adherence of granulocytes from young andaged rats at any time point tested.

3.5. The effect of PYY on phagocytosis and H2O2 production

Granulocytes from young rats responded better to stimulationwith zymosan (125 μg/ml) when compared to granulocytes from

Fig. 5. (A) The effect of PYY (10−12–10−6 M) in vitro stimulation on granulocyte adherence capacity in adult rats (8 months) after 10 min, 20 min and 30 min. (B) A specificantagonist of Y1 receptor, BIBO 3304 (10−6 M), antagonized PYY (10−6 M)-induced increase in adherence of granulocyte from adult rats after 20 min. (C) The in vitro effect of PYY(10−6 M) on adherence of granulocytes from young (3 months), adult (8 months) and aged (24 months) rats. The number of rats per group was eight. Statistically significantdifferences: ⁎pb0.05, PYY vs. corresponding MEM, and BIBO 3304 vs. MEM; and apb0.01, PYY+BIBO 3304 vs. PYY.

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adult and aged rats, indicating that aging is associated with reducedphagocytosis capability (Fig. 6A). PYY (20 μg), directly injected intothe air-pouch with carrageenan did not influence phagocytosis ofzymosan in granulocytes obtained from young rats but diminished itin granulocytes from adult and aged rats (Fig. 6A). Following the invitro stimulation with 25 nM PMA, granulocytes from aged ratsproduced more H2O2 in comparison with granulocytes obtained fromadult and aged rats (Fig. 6B). Furthermore, PYY directly injected intothe air-pouch decreased the PMA-stimulated H2O2 production ingranulocytes regardless of age, and lessen the H2O2 concentration inair-pouch exudate in young and adult rats (insert in Fig. 6B).

When applied in vitro, PYY decreased phagocytosis of zymosan ingranulocytes from young (10−6 M) and adult (10−8 M and 10−6 M),but not from aged rats, in a dose-dependent manner (Fig. 6C).Additionally, PYY in vitro modulated the H2O2 production ingranulocytes from young and adult rats (Fig. 6D). Specifically, at allconcentrations tested, PYY increased the PMA-stimulated granulocyteH2O2 production in young rats, except when used at a concentrationof 10−10 M, which caused a decrease in H2O2 production. Ingranulocytes obtained from adult rats, PYY slightly increased H2O2

production at 10−12 M.

3.6. The effects of NPY-related peptides and Y receptor antagonists onphagocytosis and H2O2 production

Similarly to PYY, LP-NPY (Y1/Y5 receptor agonist) significantlysuppressed zymosan phagocytosis in granulocytes from adult rats at10−8 M and 10−6 M, while NPY3–36 (Y2/Y5 receptor agonist) had no

effect at any concentration tested (Fig.7A). These findings suggestedthe role for Y1 receptor in the suppression of phagocytosis by PYY.Indeed, the suppressive effect of PYY (10−8 M) was antagonized byY1 receptor antagonist BIBO 3304 (10−6 M), and not by Y2 receptorantagonist BIIE 0246 (10−6 M) (Fig. 7B). However, the DP4 inhibitorIleThia (10−6 M) was unable to intensify the suppressive effect of PYYon granulocyte phagocytosis.

NPY-related peptides decreased H2O2 production in granulocytesfrom adult rats, and NPY3–36 appeared to be more efficient comparedto LP-NPY (Fig. 8A). Although the Y2 receptor involvement wasanticipated, a specific Y2 receptor antagonist, BIIE 0246 (10−6 M),was unable to block the suppressive effect of PYY (10−10 M), while itfurther decreased the H2O2 production (Fig. 8B). Besides, BIIE 0246,IleThia and BIBO 3304 (10−6 M) decreased the PMA-stimulatedgranulocyte H2O2 production to a similar extent as PYY (10−10 M).

4. Discussion

It is well known that most DP4/CD26 activity is membrane-expressed, mainly located on the vasculature/endothelial cells and onhepatocytes [37,38]. There is, however, a strong circulating DP4-likeactivity. Although debate remains as to the relative contributions ofcleaved membrane CD26 to the soluble circulating DP4-like enzy-matic activity, the functional consequences, i.e. inactivation andprevention of systemic effects of pro-inflammatory peptide media-tors, will be identical. Accordingly, the decreased plasma and exudateDP4 activity found in aged rats is consistent with the increasedaccumulation of granulocytes into the air-pouch, following the

Fig. 6. The effect of direct injection of PYY (20 μg) into the air pouch on: (A) 125 μg/ml ZYM-stimulated phagocytosis, and (B) 25 nM PMA-stimulated H2O2 production ofgranulocytes from young (3 months), adult (8 months) and aged (24 months) rats 24 h after challenge with carrageenan (20 mg). The insert shown in (B) represents the H2O2

concentration in air-pouch exudates. The effect of PYY (10−12–10−6 M) in vitro on: (C) 125 μg/ml ZYM-stimulated phagocytosis, and (D) 25 nM PMA-stimulated H2O2 production ofgranulocytes from young (3 months), adult (8 months) and aged (24 months) rats. The number of rats per group was eight. Statistically significant differences: ⁎pb0.05, ⁎⁎pb0.01,⁎⁎⁎pb0.001, and ⁎⁎⁎⁎pb0.0001 PYY vs. corresponding Control, PMA or ZYM; and apb0.01 young vs. adult and aged; bpb0.0001 aged vs. young and adult, and cpb0.05, young vs.adult and aged.

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carrageenan injection. The changes of DP4 activity in rats due to agingappear to be strain specific as F344 rats, aged from 1 month to12 months, exhibited equal serum DP4 activity level [39]. Thediminished DP4-like activity in plasma and air-pouch exudates ofaged rats is in agreement with age-related decrease of DP4 activitypreviously reported in humans [40] and rats [14].

The adhesion of leukocytes to endothelial cells is an important stepleading to the migration of inflammatory cells from the circulation totissue sites of infection. In humans, aging has been related to normal[41–43], increased [44] and decreased [45,46] adherence ability ofneutrophils. Our results clearly showed that the expression of theadhesive molecule CD11b on granulocytes was insufficiently in-creased in aged rats to improve their adherence capacity. Despite amodest decline in adherence capacity observed for granulocytes ofaged rats, the carageenan-induced granulocytes recruitment wasmore intense in aged rats when compared to adult and young rats.

However, in adult rats, the increased adherence capacity ofgranulocytes following the in vitro treatment with PYY was inaccordance with slightly elevated accumulation of granulocytes intothe air-pouch after the treatment with PYY in vivo. In addition, theantagonistic in vitro action of BIBO3304 suggested a role for Y1receptor in PYY-induced enhancement of granulocyte adherence.Furthermore, NPY3–36, which is devoid of Y1 receptor specificity, did

not modulate granulocyte adherence capacity in vitro (data notshown). A stimulatory effect of NPY on adherence of leukocytes fromspleen, axillary nodes and thymus, as well as its inhibitory effect oncells from peritoneum in young, but not in aged mice has beendemonstrated [12,19,20]. The low expression level of Y1 receptors onair-pouch exudate cells could account for the absence of the Y1receptor-mediated stimulating effect of PYY on granulocyte ad-herence capacity in young and aged rats. Quite the opposite, a de-creased recruitment of inflammatory cells following the in vivotreatment with PYY observed in aged rats, probably involved a certainage-associated pattern of expression of other molecules apart fromthe NPY Y1 receptor. Furthermore, the anti-inflammatory effect ofPYY in vivo may implicate its action via Y1, Y2 and/or Y5 receptorsubtypes expressed on vascular endothelial cells [32,47].

The current results for the first time demonstrate that theprevalence of Y1, Y2 and Y5 receptor subtypes on rat granulocyteschange with age. This finding opens the door for the proposed role ofthese molecules in modulation of granulocyte functions such asadherence, phagocytosis and H2O2 production. The presence of Yreceptors on human neutrophils has been recently confirmed, as wellas the involvement of Y1 and Y2 receptors in the phagocytosis and Y5receptor in the release of reactive oxygen species [18]. Our finding ofdecreased zymosan phagocytosis of granulocytes from adult and aged

Fig. 7. The in vitro effect of: (A) Y1,5 receptor agonist LP-NPY (10−12–10−6 M) and Y2,5receptor agonist NPY3–36 (10−12–10−6 M); and (B) an inhibitor of DP4, IleThia (10−6 M),Y1 receptor antagonist BIBO3304(10−6 M)andY2 receptor antagonist BIIE 0246 (10−6 M)in absence or presence of PYY (10−8 M) on 125 μg/ml ZYM-stimulated phagocytosis ofgranulocytes from adult (8 months) rats. The number of rats per group was eight. Statis-tically significant differences: ⁎⁎pb0.01, vs. corresponding ZYM; and apb0.05 vs. PYY/ZYM.

Fig. 8. The in vitro effect of: (A) Y1,5 receptor agonist LP-NPY (10−12–10−6 M) and Y2,5receptor agonist NPY3–36 (10−12–10−6 M); and (B) an inhibitor of DP4, IleThia (10−6 M),Y1 receptor antagonist BIBO3304 (10−6 M)andY2 receptor antagonist BIIE 0246 (10−6 M)in absence or presence of PYY (10−10 M) on 25 nM PMA-stimulated H2O2 production ofgranulocytes from young (3 months) rats. The number of rats per group was eight.Statistically significant differences: ⁎pb0.05, ⁎⁎pb0.01, and ⁎⁎⁎pb0.001, vs. PMA, andbpb0.001, vs. PYY/PMA.

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rats is in agreement with previously reported inability of phagocytesto engulf microbes in advanced age [29,48]. As the phagocytosis ofzymosan is predominantly a complement receptor-mediated process[33], the reduced phagocytosis at normal level of CD11b expression ongranulocytes — as found in aged rats, suggested an altered com-plement receptor signaling, as it has been proposed in humans [28].The intracellular signaling events following the ligand binding forspecific receptor can bemodified by changes in themembrane fluidityseen with age, which explains the decreased functionality ofneutrophils in the elderly [49]. Interestingly, the anti-inflammatoryeffect of PYY in vivo was indicated by suppression of phagocytosis inadult and aged, but not in young rats. Since the suppression ofphagocytosis is Y1 receptor-mediated effect of NPY-related peptides,as shown earlier [17] and confirmed in this study, the absence of theeffect of PYY injected directly into the air-pouch in young rats could beattributed to their high DP4-like activity in plasma and air-pouchexudates. It should be noted that PYY decreased zymosan phagocy-tosis in granulocytes of young and adult rats in vitro, which is clearlyindependent of plasma DP4-like activity, and most likely unaffected

by minute DP4-like activity in air-pouch exudate cells, i.e., 10 timeslower from the activity previously detected in rat peritonealmacrophages [14].

Although lower concentrations of H2O2 found in air-pouchexudates of aged rats indicated that H2O2 production in vivo declinedwith aging, granulocytes from aged rats robustly responded to the invitro stimulation with PMA that directly activated PKC and stimulatedoxidative burst without phagocytosis. There were no age-relateddifferences in the suppressive effect of PYY on granulocyte H2O2

production following the PMA-stimulation in vitro, when PYY wasinjected directly into the air-pouch. Moreover, the suppression ofH2O2 production by PYY in vivo was independent of DP4-like activity.In contrast, a bimodal dose-dependent effect of PYY in vitro on H2O2

production was observed in granulocytes from young rats only. Theinability of Y1 receptor antagonist (BIBO 3304) to prevent the PYY-induced decrease of granulocyte H2O2 production, excluded the rolefor Y1 receptor in this suppression. Y2 receptor antagonist (BIIE 0246)in vitro was also unsuccessful in blocking the PYY-induced decreaseof granulocyte H2O2 production. Because Y1,5 (LP-NPY) and Y2,5(NPY3–36) receptor agonists in vitro reduced granulocyte H2O2 pro-duction in young rats, the participation of Y5 receptor in the sup-pressive effect of NPY-related peptides on H2O2 production was

108 M. Dimitrijević et al. / Regulatory Peptides 159 (2010) 100–109

suggested. Results of the present study are in accordance with ourearlier reports showing that Y5 receptor is responsible for suppressionof oxidative burst dependent on PKC activity in both granulocytes [17]and macrophages [13]. Y1 receptor attenuated the oxidative burst ingranulocytes initiated after phagocytosis of zymosan that is depen-dent on binding of zymosan for complement receptor CR3 [17]. Itshould be noted that IleThia (DP4 inhibitor) and Y receptorantagonists (BIBO 3304 and BIIE 0246) exerted suppressive-likeeffects on granulocyte H2O2 production itself. Similar results havebeen previously reported for rat peritoneal macrophages [13]. There isa possibility that the PMA-stimulated granulocytes released PYY orNPY that tonically regulated oxidative burst via Y5 receptors. Other-wise, a receptor non-specific suppression or modulation of oxidativeburst by some other endogenous peptide(s) released from the PMA-stimulated granulocytes could be implicated.

The difference between in vivo and in vitro effects of PYY ongranulocyte functions in aged rats suggested that PYY affected theenvironment (the hormone/cytokine milieu) in which the cell residedin vivo and indirectly caused suppression of granulocyte functions.The failure of PYY to modulate granulocyte functions following the invitro treatment of cells from aged rats could be a consequence ofaltered Y receptor functioning. Altered receptiveness for endogenouspeptides, such as vasoactive kinins, due to aging has been demon-strated in rats [50].

Recently, several studies using different rat strains showed thatthe basal levels of NPY and PYY in plasma or sera were at the order ofmagnitude of femtomoles [31,51,52] and did not change significantlydue to aging. However, the dose of PYY employed in our study was atmicromoles level (20 μg of PYY in 2 ml of carrageenan), and thus signi-ficantly above the reported physiological levels of PYY in rat sera.Therefore, it is unlikely that naturally occurring PYY interfered withthe pharmacological effects of PYY on granulocyte functions followingthe in vivo treatment.

In summary, our study shows that PYY modulates different ratgranulocyte functions, in an age-dependant manner. As a novelfinding, we report here the presence of different Y receptor subtypes,mediating the PYY actions, on rat granulocytes. Aging is associatedwith impaired response of granulocytes to PYY in vitro probably dueto lower Y1/Y2/Y5 receptors expression. However, the effect of PYY ongranulocytes microenvironment together with aging-induced de-crease in plasma DP4 activity is responsible for prominent anti-inflammatory action of PYY in vivo in aged rats, emphasizing amultifaceted regulation of anti-inflammatory activity of NPY and NPY-related peptides with respect to age.

Acknowledgements

This workwas supported by grant from theMinistry of Science andTechnological Development, Belgrade, Serbia (145049).

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