Journal Identification = PEP Article Identification = 68381 Date: July 8, 2011 Time: 3:18 pm

Prm

KAZa

b

c

d

e

Yf

a

ARRAA

KAWEMIL

1

nawia

S

0d

Peptides 32 (2011) 1439–1446

Contents lists available at ScienceDirect

Peptides

j ourna l ho me pa ge: www.elsev ier .com/ locate /pept ides

ituitary adenylate cyclase-activating polypeptide plays an anti-inflammatoryole in endotoxin-induced airway inflammation: In vivo study with gene-deletedice

risztian Elekesa, Katalin Sandora, Andras Moricza, Laszlo Kereskaib, Agnes Kemenya, Eva Szokea,niko Perkecza, Dora Reglodic, Hitoshi Hashimotod,e,f, Erika Pintera, Janos Szolcsanyia,suzsanna Helyesa,∗

Department of Pharmacology and Pharmacotherapy, Faculty of Medicine, University of Pecs, Szigeti Str. 12., H-7624 Pecs, HungaryDepartment of Pathology, Faculty of Medicine, University of Pecs, Szigeti Str. 12., H-7624 Pecs, HungaryDepartment of Anatomy, Faculty of Medicine, University of Pecs, Szigeti Str. 12., H-7624 Pecs, HungaryLaboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University, Yamadaoka, Suita, Osaka 565-0871, JapanCenter for Child Mental Development, United Graduate School of Child Development, Osaka University, Kanazawa University and Hamamatsu University School of Medicine,amadaoka, Suita, Osaka 565-0871, JapanDepartment of Molecular Pharmaceutical Science, Graduate School of Medicine, Osaka University, Yamadaoka, Suita, Osaka 565-0871, Japan

r t i c l e i n f o

rticle history:eceived 9 March 2011eceived in revised form 6 May 2011ccepted 6 May 2011vailable online 14 May 2011

eywords:irway hyperresponsivenesshole body plethysmography

nhanced pause (Penh)yeloperoxidase activity

nterleukin-1�ipopolysaccharide

a b s t r a c t

The presence of pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptors in capsaicin-sensitive peptidergic sensory nerves, inflammatory and immune cells suggest its involvement ininflammation. However, data on its role in different inflammatory processes are contradictory and there islittle known about its functions in the airways. Therefore, our aim was to examine intranasal endotoxin-induced subacute airway inflammation in PACAP gene-deficient (PACAP−/−) and wild-type (PACAP+/+)mice. Airway responsiveness to inhaled carbachol was determined in unrestrained mice with wholebody plethysmography 6 h and 24 h after LPS. Myeloperoxidase (MPO) activity referring to the number ofaccumulated neutrophils and macrophages was measured with spectrophotometry and interleukin-1�(IL-1�) concentration with ELISA from the lung homogenates. Histological evaluation and semiquan-titative scoring were also performed. Bronchial responsiveness, as well as IL-1� concentration andMPO activity markedly increased at both timepoints. Perivascular edema dominated the histologicalpicture at 6 h, while remarkable peribronchial granulocyte accumulation, macrophage infiltration and

−/−

goblet cell hyperplasia were seen at 24 h. In PACAP mice, airway hyperreactivity was significantlyhigher 24 h after LPS and inflammatory histopathological changes were more severe at both timepoints.MPO increase was almost double in PACAP−/− mice compared to the wild-types at 6 h. In contrast,there was no difference between the IL-1� concentrations of the PACAP+/+ and PACAP−/− mice. Theseresults provide evidence for a protective role for PACAP in endotoxin-induced airway inflammation andhyperreactivity.

. Introduction

Endotoxin (lipopolysaccharide: LPS) is a constituent of Gram-egative bacteria found in the environment including house dust,nd it induces an intensive inflammatory reaction in the air-

ays accompanied by bronchopulmonary hyperreactivity. This

s related to an increased responsiveness to bronchoconstrictorgents, e.g. muscarinic receptor agonists [32]. Intranasal endotoxin

∗ Corresponding author at: Department of Pharmacology, University of Pecs,zigeti u. 12., H-7624 Pecs, Hungary. Tel.: +36 72 536001/5591; fax: +36 72 536218.

E-mail address: zsuzsanna.helyes@aok.pte.hu (Z. Helyes).

196-9781/$ – see front matter © 2011 Elsevier Inc. All rights reserved.oi:10.1016/j.peptides.2011.05.008

© 2011 Elsevier Inc. All rights reserved.

administration in mice evokes an acute pulmonary inflammationwith a well-described mechanism involving neutrophil infiltra-tion and cytokine, mainly TNF-� and IL-1�, production [49,51].Granulocyte activation, as opposed to recruitment alone, is animportant factor of LPS-induced inflammation and consequentbronchial hyperreactivity, since their depletion is fully suppres-sive [50]. They are recruited to the airway epithelial/subepithelialregions and cause tissue damage via the production and releaseof oxygen radicals, proteases, cytokines and chemokines [30],

which attract and stimulate mononuclear cells and lympho-cytes.

Our earlier data demonstrated that pro-inflammatory sen-sory neuropeptides, substance P (SP) and calcitonin gene-related

Journal Identification = PEP Article Identification = 68381 Date: July 8, 2011 Time: 3:18 pm

1 ides 32

pnaotaatnai(ttsfi[

ciGlshtmeonarnpsp

wmmaifttct[codhb

2

2

owmclfo

440 K. Elekes et al. / Pept

eptide (CGRP), are released from capsaicin-sensitive sensoryerve terminals of the lung in response to endotoxin-inducedirway inflammation and they contribute to the developmentf the inflammatory reaction and hyperresponsiveness [13]. Fur-hermore, we have shown that in this model somatostatin islso released from these fibers and mediate potent inhibitoryctions through the activation of somatostatin receptor sub-ype 4 (sst4) [22,23]. These data provided clear evidence thateuropeptides released from sensory nerves can both positivelynd negatively modulate this type of inflammatory reactionn the lung. Pituitary adenylate cyclase-activating polypeptidePACAP), which is a member of the vasoactive intestinal pep-ide (VIP)/secretin/glucagon peptide family, is also present inhe cell bodies [40,42] and peripheral terminals of capsaicin-ensitive primary sensory neurons [14,69]. In most sensorybers PACAP coexists with CGRP [14,19,26,41,53,54] and SP39,53,57].

PACAP exerts several functions in a large array of physiologi-al and pathophysiological processes related to inflammation andmmune responses by activating three receptors [61]. These are

protein-coupled receptors mainly associated with the adeny-ate cyclase and phospholipase C: the PAC1 receptor whichpecifically binds PACAP and the VPAC1/VPAC2 receptors whichave a similar binding affinity for PACAP and VIP. Both recep-ors have been described, among others, on neurons, smooth

uscle cells and several inflammatory cells [10,55,61,70]. Ourarlier data provided evidence that PACAP inhibits the releasef SP and CGRP from peripheral terminals of capsaicin-sensitiveerves of isolated rat tracheae [43], as well as acute neurogenicnd non-neurogenic inflammatory processes in both mice andats [22,43]. These inhibitory actions on nerve terminals can-ot be explained by the presently known signal transductionathways, therefore, the existence of a yet unidentified receptor,plice variant and/or intracellular signaling process can be pro-osed.

In a recent study, increased PAC1 receptor mRNA expressionas found in the mouse lung in ovalbumine-induced inflam-ation. In this chronic asthma model PAC1 receptor-deficientice showed markedly increased inflammatory reactions, while

PAC1-selective agonist treatment resulted in significant anti-nflammatory effects. However, there was no effect on lungunction [31]. PACAP deficient mice provide a tool to study func-ions of endogenous PACAP. PACAP deficient mice have been showno display several abnormalities, such as behavioral alterations,hanged sympathetic nerve activity and increased vulnerabilityo ischemia/reperfusion injury in the small intestine and kidney16,20,21,58]. In a dextran-induced model of colitis, more severelinical symptoms as well as increased inflammation have beenbserved [44]. The aim of the present study was to investigate theevelopment of endotoxin-induced pneumonitis and consequentyperresponsiveness in PACAP-deficient mice using functional,iochemical, immunological and morphological techniques.

. Materials and methods

.1. Animals

Experiments were performed on female PACAP gene knock-ut mice (PACAP−/− [20]) and their female wild-type counterpartseighing 20–25 g, which were bred and kept in the Laboratory Ani-al Center of the University of Pécs under standard pathogen free

onditions at 24–25 ◦C provided with standard chow and water adibitum. Each experimental series presented in this paper were per-ormed in blocks with 6–8 mice per day. Each group presented inne figure ran at the same time.

(2011) 1439–1446

2.2. Ethics

All experimental procedures were carried out according to the1998/XXVIII Act of the Hungarian Parliament on Animal Protec-tion and Consideration Decree of Scientific Procedures of AnimalExperiments (243/1988) and complied with the recommendationsof the Helsinki Declaration. The studies were approved by the EthicsCommittee on Animal Research of Pécs University according to theEthical Codex of Animal Experiments and license was given (licenseNo.: BA 02/200-6-2001).

2.3. Endotoxin-induced airway inflammation model

Subacute interstitial lung inflammation was evoked in mice(n = 8–10 per group) by intranasal administration of 60 �lEscherichia coli (serotype: 083) lipopolysaccharide (LPS; 167 �g/ml;Sigma, St. Louis, MO, USA) dissolved in sterile PBS. The same volumeof sterile PBS was applied to control mice regarded as intact (non-inflamed) animals. This procedure was performed in light etheranesthesia by gently holding the mouse in the left hand in a verticalposition and the solution was dropped on the nose with a pipettein one portion. It was completely sniffed in within approximately20 s, but the animal was held in the same vertical position for aminute to provide a relatively equal distribution of the solution inthe lung. Airway reactivity measurements were done 6 and 24 hfollowing the challenge [22,23,46]. This type of pneumonitis is awell-established model with mostly identified mechanisms: LPSbinds to a specific LPS binding protein (LBP) forming a complexthat activates the CD14/TLR4 receptor structure predominantly onmacrophages triggering the production of a variety of inflammatorymediators, which leads to a remarkable neutrophil activation [38].Data showing that intranasal administration of this LPS dose evokesmaximal inflammation (neutrophil accumulation and inflamma-tory cytokine production) 24 h after its instillation served as basisfor choosing the latter timepoint, the development of the inflam-matory process was aimed to be analyzed at 6 h.

2.4. Assessment of bronchoconstriction

Airway responsiveness in conscious, spontaneously breathinganimals was measured by recording respiratory pressure curvesby whole body plethysmography (Buxco Europe Ltd, Winchester,UK) [11,37]. Aerosolized saline and then the muscarinic acetyl-choline receptor agonist carbachol (carbamoyl-choline; Sigma, St.Louis, MO, USA) were administered in increasing concentrations(50 �l solution of the 11 and 22 mM concentrations nebulized intothe chamber for each mouse for 50 s) to induce bronchoconstric-tion 6 and 24 h after LPS. Recordings were taken and averagedfor 15 minutes following each nebulization [17]. Baseline val-ues usually returned at the end of this period. Enhanced pause(Penh) was measured as an indicator of bronchoconstriction andconsequent increase of airway resistance. Penh is a complex, cal-culated parameter ((expiratory time/relaxation time) − 1): (max.expiratory flow/max. inspiratory flow), which closely correlateswith airway resistance measured by traditional invasive tech-niques as evidenced by our own studies [13,22,23], as well asby other authors [11,37]. Percentage increases of the mean Penhvalues above baseline after each carbachol stimulation were calcu-lated.

2.5. Histological studies and scoring

At the end of the experiments the animals were sacrificed bycervical dislocation under ketamin (100 mg/kg i.p.; Richter-GedeonLtd., Budapest, Hungary) and xylazine (10 mg/kg i.m.; Phylaxia-Sanofi, Veterinary Biology Co. Ltd., Budapest, Hungary) anesthesia,

Journal Identification = PEP Article Identification = 68381 Date: July 8, 2011 Time: 3:18 pm

ides 32 (2011) 1439–1446 1441

tieaeSmspd

2

acpqavpw

2

t(iss(mwaiptnoom1la1wcdvt

2

nhbfstfbppb4r

Fig. 1. Bronchial hyperreactivity to inhaled carbachol. LPS-induced bronchialhyperreactivity measured in freely moving, unrestrained mice by whole bodyplethysmography. Bronchoconstriction was evoked by aerosolization of 11 and22 mM carbachol (50 �l/mouse) and the responses were evaluated by percentageincrease of the mean Penh above baseline for a 15 min period after each carba-chol stimulation. Each value represents the means ± S.E.M. of n = 8–10 experiments;*p < 0.05 vs. LPS-treated PACAP+/+ mice and +p < 0.05 vs. saline-treated respective

K. Elekes et al. / Pept

he lungs were excised and the left lung samples were fixedn 4% formaldehyde for 8 h for histological processing. Paraffin-mbedded specimens were sectioned with microtome (5–7 �m)nd stained with hematoxylin (Sigma, St. Louis, MO, USA) andosin (Molar Chemicals Ltd., Budapest, Hungary) or periodic acid-chiff (PAS; Sigma, St. Louis, MO, USA) to more precisely visualizeucus producing goblet cells. Evaluation and semiquantitative

coring of the inflammatory changes at the 24 h time-point waserformed by an expert pathologist blinded from the experimentalesign.

.5.1. Quantitative evaluation of the perivascular edemaAt the 6 h time-point the predominant histopathological alter-

tion was the perivascular edema and diffuse, initial inflammatoryell infiltration, therefore, semiquantitative scoring could not beerformed. The remarkable perivascular edema formation wasuantified by drawing the contours of these edematous regionst the 100× magnification in 4 distinct randomly chosen fields ofision in each slide with the help of the “Analysis” software. Thisrogram automatically calculates the edema region values, whichere added for each histological section.

.5.2. Scoring of the LPS-induced acute inflammatory reactionAt the 24 h time-point scoring parameters were chosen on

he basis of the presence or abundance of the following [68]:1) perivascular edema (0: absent; 1: mild to moderate, involv-ng less than 25% of the perivascular spaces; 2: moderate toevere, involving more than 25% but less than 75% of perivascularpaces; 3: severe involving more than 75% of perivascular spaces);2) perivascular/peribronchial acute inflammation (0: absent; 1:

ild acute inflammation in the perivascular edematous spaceith fewer than 5 neutrophils per high-power field; 2: moder-

te acute inflammation in the perivascular spaces extending tonvolve the peribronchial spaces with more than 5 neutrophilser high power field in these regions; 3: severe acute inflamma-ion in the perivascular and peribronchial spaces with numerouseutrophils around most bronchioles); (3) goblet cell metaplasiaf the bronchioles (0: absent; 1: few goblet cells present in oner two bronchioles; 2: large number of goblet cells present); (4)acrophages/mononuclear cells in the alveolar spaces (0: absent;

: present in fewer than 25% of alveolar spaces, 2: >25% of alveo-ar spaces). The score values for these individual parameters weredded to form a composite inflammatory score ranging from 0 to0. From every specimen (8–10 mice in each group) 4–5 sectionsere taken from different depths to give a representative appre-

iation of the whole lung. Mean scores were determined from theifferent sections of the individual animals and composite scorealues of the different experimental groups were calculated fromhese mean scores [13,22].

.6. Measurement of myeloperoxidase (MPO) activity in the lung

The LPS-induced inflammatory response is a predominantlyeutrophilic inflammation, they represent the major cell type in theistological picture together with macrophages [32,33,38]. Sinceoth cells express the MPO enzyme, its activity was determinedrom the lung homogenates, as a biochemical marker of the inten-ity of the inflammatory reaction. The right lungs were cut intowo parts, and after measuring the weight of the pieces, one wasrozen at −20 ◦C for later determination of neutrophil accumulationy spectrophotometric determination of the MPO activity. Uponrocessing, these samples were thawed and chopped into small

ieces then homogenized in 4 ml 20 mM potassium-phosphateuffer (pH 7.4). The homogenate was centrifuged at 10,000 × g at◦C for 10 min and supernatant was removed. The pellet was then

esuspended in 4 ml 50 mM potassium-phosphate buffer contain-

non-inflamed group (one-way ANOVA followed by Bonferroni’s modified t-test).

ing 0.5% hexadecyl-trimethyl-ammonium-bromide (pH 6.0) andcentrifuged again. The spectrophotometric measurement was donefrom the supernatant using H2O2-3,3′,5,5′-tetramethyl-benzidine(TMB/H2O2). Reactions were performed in 96-well microtitreplates in room temperature. The optical density (OD) at 620 nm wasmeasured at the 0 and 5 min timepoints using a microplate reader(Labsystems) and plotted. The reaction rate was determined as�OD/min according to the slope of the line. A calibration curve wasthen created with the rate of reaction plotted against the humanstandard MPO preparation [22,23]. All reagents used for this assaywere obtained from Sigma, St. Louis, MO, USA.

2.7. Measurement of interleukin-1 ̌ (IL-1ˇ) and tumor necrosisfactor- ̨ (TNF-˛) concentration in the lung

The other half of each right lung was frozen separately inliquid nitrogen and stored at −80 ◦C. The wet weight of the sam-ples was measured before homogenization in 450 �l RPMI 1640buffer (Biochrom Ltd., Berlin, Germany) containing 50 �l phenyl-methyl-sulphonyl-fluoride (PMSF; Sigma, St. Louis, MO, USA) witha polytrone homogenizer (Kika Lab Techniques) at 13,500 rpm for2 min. The homogenates were centrifuged for 10 min at 10,000 rpmand 4 ◦C. The concentrations of the inflammatory cytokines IL-1�and TNF-� were determined by specific sandwich ELISA techniques(BD Biosciences, NJ, USA).

2.8. Statistical analysis

Values for all measurements were expressed as the mean ± SEMof n = 8–10 mice in each group. Evaluation of the mean Penh datahas been performed by one-way ANOVA followed by Bonferroni’smodified t-test to see statistical differences between different datasets and then between the respective data points. Histological

inflammatory score values were analyzed with Kruskal–Wallis fol-lowed by Dunn’s post test.

Journal Identification = PEP Article Identification = 68381 Date: July 8, 2011 Time: 3:18 pm

1442 K. Elekes et al. / Peptides 32 (2011) 1439–1446

Fig. 2. Representative histopathological pictures of the lung obtained from PACAP+/+ and PACAP−/− mice. Histopathological pictures of lung samples obtained (A) 24 h afterintranasal saline treatment (non-inflamed control); (B) 6 h following LPS administration; and (C) 24 h after LPS-treatment of PACAP+/+ mice. In comparison, the lower panelsshow the respective pictures of PACAP−/− mouse lung samples: (D) saline-treatment; (E) 6 h after LPS-treatment; (F) 24 h after LPS-administration. Double headed arrows inpanels B, C, E and F indicate the perivascular edema formation. Panels G–H represent higher magnifications of the representative histopathological alterations: perivasculare a, whw ow). Sm in pa

3

3P

cPttdaamopoP

3m

t

dema with infiltrating inflammatory cells (G: double-headed arrow indicates edemhite arrow indicates a macrophage) and mucus-producing goblet cells (I, black arragnifications; a: alveolar spaces, b: bronchi, v: vessels. Scale bars indicate 200 �m

. Results

.1. Inflammatory airway hyperresponsiveness in PACAP+/+ andACAP−/− mice

Inhalation of the muscarinic receptor agonist carbachol evokedoncentration-dependent bronchoconstriction as shown by theenh curves. These responses were markedly enhanced in the LPS-reated groups at both the 6 h and 24 h time-points compared tohe non-inflamed saline-treated controls. There was no significantifference between the carbachol-induced responses of PACAP+/+

nd PACAP−/− mice either without LPS treatment or 6 h after LPSdministration. In contrast, 24 h after the induction of the inflam-ation both the maximal Penh values were higher and the duration

f the responses were longer in PACAP gene knockout mice, theercentage increase of the Penh responses above baseline in casef the 22 mM carbachol stimulation was significantly higher in theACAP−/− group than in the PACAP+/+ one (Fig. 1).

.2. Inflammatory changes in the lung of PACAP+/+ and PACAP−/−

ice

Histological examination and scoring revealed that comparedo the intact lung structure (Fig. 2A and D), LPS induced a promi-

ite arrow shows a macrophage; H: black arrow points to a neutrophil granulocyte,amples were stained with PAS and shown at 200× (A–F), 400× (G–H) and 600× (I)nels A–F and 50 �m in panels G–I.

nent perivascular edema formation with initial diffuse granulocyteinfiltration 6 h after its administration (Fig. 2B and E). The perivas-cular edema was markedly greater in the PACAP−/− group (Fig. 2E),this significant difference could also be justified with the quan-titative analysis. The total area of the edematous tissue in 4randomly chosen fields of vision at 100× magnification was about4–5-times higher in the PACAP−/− animals (Fig. 3A). At the 24 htime-point peribronchial/perivascular edema (Fig. 2G) was stillpresent, but decreased compared to that observed at 6 h. Further-more, granulocyte accumulation around the bronchi, infiltrationof mononuclear cells (Fig. 2H) and hyperplasia of mucus pro-ducing goblet cells (Fig. 2I) were also detected (Fig. 2C and F).The composite inflammation score determined on the basis ofthese inflammatory parameters was significantly higher in PACAPreceptor gene-deficient mice than in their wild-type counterparts(Figs. 2C, F, 3B).

3.3. Myeloperoxidase activity in the lung of PACAP+/+ andPACAP−/− mice

The basal MPO activity measured in the non-inflamed lunghomogenates of saline-treated control mice did not differ signif-icantly between the PACAP+/+ and PACAP−/− groups. Endotoxinadministration induced an about 2–3-fold increase of MPO activity

Journal Identification = PEP Article Identification = 68381 Date: July 8, 2011 Time: 3:18 pm

K. Elekes et al. / Peptides 32 (2011) 1439–1446 1443

Fig. 3. Histopathological evaluation of LPS-induced inflammatory changes in the lung of PACAP+/+ and PACAP−/− mice. Panel A shows the total area of edema around the vesselsand the bronchioles 6 h after intranasal LPS administration. The lower panel (B) represents the (a) detailed and the (b) composite semiquantitative inflammation score valuesdetermined 24 h after the induction of the inflammation. The parameters for scoring were perivascular edema formation (edema), perivascular/peribronchial granulocytea d gobln 8–10

mlP

3

ssliba

4

cisp

ccumulation (gran.), infiltration of macrophages into the alveolar spaces (mac.) an = 6–8 mice, boxes represent the median with the upper and lower quartiles of n =

easured in the lung homogenates of wild-type mice 6 h and 24 hater. This elevation was significantly higher, about 4-fold in theACAP knockout group at the earlier time-point (Fig. 4).

.4. Interleukin-1 ̌ and TNF- ̨ concentrations in the lung

Inflammatory cytokine concentrations in the non-inflamed lungamples of saline-treated PACAP+/+ and PACAP−/− mice were veryimilar, about 2 ng/g wet tissue for IL-1� and around the detectionimit for TNF-�, respectively. The levels of these cytokines signif-cantly increased the 6 h and the 24 h time-points in both groups,ut there were no remarkable differences in the PACAP-deficientnimals compared to the wildtypes (Fig. 5).

. Discussion

The present results provide clear morphological and bio-

hemical evidence that endotoxin-induced subacute airwaynflammation is more severe in PACAP-deficient mice. There is aignificantly greater perivascular edema formation at the 6 h time-oint and a higher histopathological inflammatory score at 24 h.

et cell hyperplasia in the bronchioles (goblet). Columns show the means ± S.E.M. ofanimals; *p < 0.05, **p < 0.01 vs. PACAP+/+ (unpaired t-test; Mann–Whitney U test).

Furthermore, a larger number of accumulated granulocytes can bedetected in the early stage in case of PACAP deficiency as shown bygreater the MPO activity and also by the histopathological exami-nation. In contrast to these results, the concentrations of the twogeneral inflammatory cytokines, IL-1� and TNF-�, synthesized byseveral inflammatory cells, such as granulocytes, macrophages andalso lymphocytes were not significantly different in the PACAP-deficient mice. These data suggest that the anti-inflammatory effectof PACAP is not mediated by inhibiting the production of thesewidely expressed cytokines, which are important, but not the pre-dominant cellular mediators of this type of inflammatory reactionat the examined timepoints. Other parameters reflecting the func-tion of these cells might be decreased by PACAP. Furthermore, thereare many lymphocytes as well, the infiltration of which is secondaryin this model, mainly induced by macrophage and neutrophil-produced mediators. They are not involved in the semiquantitativescoring, but they also synthesize both IL-1� and TNF-�. Inflamma-tory airway hyperreactivity is not altered by the lack of PACAP in the

early stage, but markedly increases later. This result suggests thatin this mouse model PACAP exerts an indirect action on bronchialhyperresponsiveness through the inhibition of the inflammatoryreaction.

Journal Identification = PEP Article Identification = 68381 Date: July 8, 2011 Time: 3:18 pm

1444 K. Elekes et al. / Peptides 32

Fig. 4. Myeloperoxidase activity in the lung samples of PACAP+/+ and PACAP−/− mice.MPO activity, as a quantitative indicator of the number of accumulated granulocytesand macrophages, was determined from homogenized lung samples 6 h and 24 hafter intranasal LPS administration. Saline-treated animals examined at both time-points served as non-inflamed controls. Each column represents the means ± S.E.M.of n = 8–10 mice; *p < 0.05 vs. respective PACAP+/+ group and +p < 0.05 and ++p < 0.01vs. respective saline-treated mice. (ANOVA followed by Bonferroni’s modified t-test.)

Fig. 5. Inflammatory cytokine concentrations of lung samples of PACAP+/+ and PACAP−/−

mice. The inflammatory cytokines (A) IL-1� and (B) TNF-� were determined fromlung homogenates 6 h and 24 h after intranasal LPS administration compared tosaline-treated animals examined at both time-points. Each column represents themeans ± S.E.M. of n = 8–10 mice; *p < 0.05, **p < 0.01 vs. respective saline-treatedmice. (ANOVA followed by Bonferroni’s modified t-test.)

(2011) 1439–1446

Immunohistochemical data demonstrate the presence of PACAPin capsaicin-sensitive sensory nerves [15], and our earlier resultsshowed that a significant amount of PACAP can be released fromthese afferent fibers of the rat tracheae in response to low concen-tration capsaicin or electrical field stimulation [43]. Furthermore,PACAP and all of its three receptors are localized in epithelial,smooth muscle and inflammatory cells in the airways of severalspecies [8,25,27,48,60]. PACAP causes relaxation of tracheal smoothmuscle [4,9,66], promotes bronchodilation [35], and increases nasalresistance in rodents [29]. It is also a potent stimulator of air-way mucus [36,62] and chloride secretion [12], suggesting its rolein airway defense. PACAP also exerts an anti-apoptotic effect inthe respiratory system and attenuates the cytotoxicity of cigarettesmoke extracts on alveolar cells [47]. Owing to the broncho-relaxant and protective properties of PACAP and VIP, syntheticanalogs have been developed for potential application in the treat-ment of asthma [5,59,67], and a VIP aerosol formulation, aviptadil, iscurrently under evaluation for the treatment of pulmonary hyper-tension [34,61].

Functional, molecular biological and immunohistochemicaldata showed that VPAC2 receptors are present on bronchialand vascular smooth muscle [3,52,56], as well as on leuko-cytes [6]. The activation of VPAC2 receptors also results in apotent vasodilator effect in the lung and prevents antigen-inducedbronchoconstriction, vasoconstriction, edema formation and gran-ulocyte accumulation in guinea pigs [45]. Furthermore, Yoshiharaand colleagues showed that both PACAP1-27 and its synthetic ana-log induced concentration-dependent smooth muscle relaxation inin vitro human [67], monkey and guinea pig bronchus preparations[66]. Another long-acting VPAC1/2 synthetic agonist protects ratalveolar cells from the cytotoxicity of cigarette smoke [47].

PACAP has been shown to modulate immune and inflamma-tory cell functions [1,18]. Some papers show its pro-inflammatoryactions, such as vasodilation and edema formation in the rabbiteye [63,64]. Intradermal injections of low PACAP-38 concentra-tions (10−14 to 10−9 M per site) evoked plasma extravasationin the rat and rabbit skin by inducing histamine release fromactivated mast cells and directly dilating blood vessels [7,65].However, the majority of the studies report on its inhibitoryactions. Intraperitoneal administration of PACAP in mice has beenshown to reduce inflammatory changes (paw swelling, erythemaand cellular response) in the collagen-induced arthritis model bymodulating several inflammatory soluble factors [2] and to ame-liorate the clinical manifestations of experimental autoimmuneencephalomyelitis by suppressing the function of antigen present-ing cells [28]. The cellular anti-inflammatory effect of PACAP canbe explained by the presently known signal transduction path-ways, since cAMP increase in the inflammatory and immune cellsleads to decreased activation, chemotaxis, phagocytosis and medi-ator/cytokine release [61]. However, we have also provided severallines of direct evidence for the ability of PACAP to inhibit therelease of SP and CGRP from capsaicin-sensitive sensory fibers ina concentration-dependent manner, as well as acute neurogenicedema formation in the skin [24]. These findings are in good cor-relation with the presently described early anti-edema action ofPACAP in the lung. In contrast to the cellular anti-inflammatoryaction of PACAP, the well-described signal transduction processesof either the PAC1 or the VPAC receptors do not serve as logicalexplanations for the observed inhibitory action at the level of thesensory nerve terminals. Therefore, the existence of a yet uniden-tified receptor, splice variant and/or intracellular signaling processcan be proposed, particularly on the afferent fibers.

The upregulation of PAC1 receptor mRNA has recentlybeen shown in the mouse lung in response to ovalbumine-induced inflammation. In this chronic asthma model PAC1receptor-deficient mice showed remarkably increased inflamma-

Journal Identification = PEP Article Identification = 68381 Date: July 8, 2011 Time: 3:18 pm

ides 32

tiedfidno

aIba

A

KTDdPe(

R

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

K. Elekes et al. / Pept

ory reactions, while a PAC1-selective agonist treatment resultedn significant anti-inflammatory effects. However, there was noffect on lung function [31]. These findings support our presentata obtained with mice deficient in PACAP, although we in factound increased hyperresponsiveness at the timepoint when thentensity of the inflammatory reaction was more pronounced. Thisifference can either be due to the different inflammatory mecha-ism or to the VPAC receptor-mediated anti-hyperreactivity effectf PACAP.

In summary, our results are the first that show that PACAP playsn important inhibitory role in LPS-induced airway inflammation.ts anti-inflammatory action is also remarkable in the early stage,ut its anti-hyperreactivity effect is presumably not a direct mech-nism, but related to the decreased inflammatory reaction.

cknowledgements

This work was supported by Hungarian Grants OTKA K72592,73044, NK78059, CNK78480, PD77474, “Science, Please! Researcheams on Innovation” programme (SROP-4.2.2/08/1/2008-0011),eveloping Competitiveness of Universities in the South Trans-anubian Region (SROP-4.2.1.B-10/2/KONV-2010-0002), Bolyaiostdoctoral Research Fellowship, as well as Grants-in-Aid for Sci-ntific Research from Japan Society for the Promotion of ScienceJSPS).

eferences

[1] Abad C, Gomariz RP, Waschek JA. Neuropeptide mimetics and antagonists inthe treatment of inflammatory disease: focus on VIP and PACAP. Curr Top MedChem 2006;6(2):151–63.

[2] Abad C, Martinez C, Leceta J, Gomariz RP, Delgado M. Pituitary adenylatecyclase-activating polypeptide inhibits collagen-induced arthritis: an exper-imental immunomodulatory therapy. J Immunol 2001;167(6):3182–9.

[3] Adamou JE, Aiyar N, Van Horn S, Elshourbagy NA. Cloning and functional charac-terization of the human vasoactive intestinal peptide (VIP)-2 receptor. BiochemBiophys Res Commun 1995;209(2):385–92.

[4] Araki N, Takagi K. Relaxant effect of pituitary adenylate cyclase-activatingpolypeptide on guinea-pig tracheal smooth muscle. Eur J Pharmacol1992;216(1):113–7.

[5] Bolin DR, Michalewsky J, Wasserman MA, O’Donnell M. Design and devel-opment of a vasoactive intestinal peptide analog as a novel therapeutic forbronchial asthma. Biopolymers 1995;37(2):57–66.

[6] Busto R, Prieto JC, Bodega G, Zapatero J, Carrero I. Immunohistochemicallocalization and distribution of VIP/PACAP receptors in human lung. Peptides2000;21(2):265–9.

[7] Cardell LO, Hjert O, Uddman R. The induction of nitric oxide-mediatedrelaxation of human isolated pulmonary arteries by PACAP. Br J Pharmacol1997;120(6):1096–100.

[8] Chatterjee TK, Sharma RV, Fisher RA. Molecular cloning of a novel variant ofthe pituitary adenylate cyclase-activating polypeptide (PACAP) receptor thatstimulates calcium influx by activation of L-type calcium channels. J Biol Chem1996;271(50):32226–32.

[9] Conroy DM, St Pierre S, Sirois P. Relaxant effects of pituitary adenylate cyclaseactivating polypeptide (PACAP) on epithelium-intact and -denuded guinea-pigtrachea: a comparison with vasoactive intestinal peptide (VIP). Neuropeptides1995;29(3):121–7.

10] Delgado M, Munoz-Elias EJ, Gomariz RP, Ganea D. Vasoactive intestinal peptideand pituitary adenylate cyclase-activating polypeptide prevent inducible nitricoxide synthase transcription in macrophages by inhibiting NF-kappa B and IFNregulatory factor 1 activation. J Immunol 1999;162(8):4685–96.

11] DeLorme MP, Moss OR. Pulmonary function assessment by whole-bodyplethysmography in restrained versus unrestrained mice. J Pharmacol ToxicolMethods 2002;47(1):1–10.

12] Derand R, Montoni A, Bulteau-Pignoux L, Janet T, Moreau B, Muller JM, et al.Activation of VPAC1 receptors by VIP and PACAP-27 in human bronchialepithelial cells induces CFTR-dependent chloride secretion. Br J Pharmacol2004;141(4):698–708.

13] Elekes K, Helyes Z, Nemeth J, Sandor K, Pozsgai G, Kereskai L, et al. Role ofcapsaicin-sensitive afferents and sensory neuropeptides in endotoxin-inducedairway inflammation and consequent bronchial hyperreactivity in the mouse.Regul Pept 2007;141(1–3):44–54.

14] Fahrenkrug J, Hannibal J. PACAP in visceral afferent nerves supplying the ratdigestive and urinary tracts. Ann N Y Acad Sci 1998;865(Dec 11):542–6.

15] Fahrenkrug J, Hannibal J. Pituitary adenylate cyclase activating polypeptideimmunoreactivity in capsaicin-sensitive nerve fibres supplying the rat urinarytract. Neuroscience 1998;83(4):1261–72.

[

[

(2011) 1439–1446 1445

16] Ferencz A, Kiss P, Weber G, Helyes Z, Shintani N, Baba A, et al. Comparison ofintestinal warm ischemic injury in PACAP knockout and wild-type mice. J MolNeurosci 2010;42(3):435–42.

17] Finney MJ, Forsberg KI. Quantification of nasal involvement in a guinea pigplethysmograph. J Appl Physiol 1994;76(4):1432–8.

18] Ganea D, Delgado M. Vasoactive intestinal peptide (VIP) and pituitary adeny-late cyclase-activating polypeptide (PACAP) as modulators of both innate andadaptive immunity. Crit Rev Oral Biol Med 2002;13(3):229–37.

19] Hannibal J, Fahrenkrug J. Pituitary adenylate cyclase-activating polypep-tide in intrinsic and extrinsic nerves of the rat pancreas. Cell Tissue Res2000;299(1):59–70.

20] Hashimoto H, Shintani N, Tanaka K, Mori W, Hirose M, Matsuda T, et al. Alteredpsychomotor behaviors in mice lacking pituitary adenylate cyclase-activatingpolypeptide (PACAP). Proc Natl Acad Sci U S A 2001;98(23):13355–60.

21] Hatanaka M, Tanida M, Shintani N, Isojima Y, Kawaguchi C, HashimotoH, et al. Lack of light-induced elevation of renal sympathetic nerve activ-ity and plasma corticosterone levels in PACAP-deficient mice. Neurosci Lett2008;444(2):153–6.

22] Helyes Z, Elekes K, Nemeth J, Pozsgai G, Sandor K, Kereskai L, et al. Roleof transient receptor potential vanilloid 1 receptors in endotoxin-inducedairway inflammation in the mouse. Am J Physiol Lung Cell Mol Physiol2007;292(5):L1173–81.

23] Helyes Z, Pinter E, Sandor K, Elekes K, Banvolgyi A, Keszthelyi D, et al. Impaireddefense mechanism against inflammation, hyperalgesia, and airway hyperre-activity in somatostatin 4 receptor gene-deleted mice. Proc Natl Acad Sci U S A2009;106(31):13088–93.

24] Helyes Z, Pozsgai G, Borzsei R, Nemeth J, Bagoly T, Mark L, et al. Inhibitory effectof PACAP-38 on acute neurogenic and non-neurogenic inflammatory processesin the rat. Peptides 2007;28(9):1847–55.

25] Hosoya M, Onda H, Ogi K, Masuda Y, Miyamoto Y, Ohtaki T, et al. Molecularcloning and functional expression of rat cDNAs encoding the receptor for pitu-itary adenylate cyclase activating polypeptide (PACAP). Biochem Biophys ResCommun 1993;194(1):133–43.

26] Ichikawa H, Sugimoto T. Pituitary adenylate cyclase-activating polypeptide-immunoreactive nerve fibers in rat and human tooth pulps. Brain Res2003;980(2):288–92.

27] Ishihara T, Shigemoto R, Mori K, Takahashi K, Nagata S. Functional expressionand tissue distribution of a novel receptor for vasoactive intestinal polypeptide.Neuron 1992;8(4):811–9.

28] Kato H, Ito A, Kawanokuchi J, Jin S, Mizuno T, Ojika K, et al. Pituitary adenylatecyclase-activating polypeptide (PACAP) ameliorates experimental autoim-mune encephalomyelitis by suppressing the functions of antigen presentingcells. Mult Scler 2004;10(6):651–9.

29] Kinhult J, Adner M, Uddman R, Cardell LO. Pituitary adenylate cyclase-activatingpolypeptide, effects in the human nose. Clin Exp Allergy 2003;33(7):942–9.

30] Kraneveld AD, Nijkamp FP. Tachykinins and neuro-immune interactions inasthma. Int Immunopharmacol 2001;1(9–10):1629–50.

31] Lauenstein HD, Quarcoo D, Plappert L, Schleh C, Nassimi M, Pilzner C,et al. Pituitary adenylate cyclase-activating peptide receptor 1 mediates anti-inflammatory effects in allergic airway inflammation in mice. Clin Exp Allergy2011;41(4):592–601.

32] Lefort J, Motreff L, Vargaftig BB. Airway administration of Escherichia coli endo-toxin to mice induces glucocorticosteroid-resistant bronchoconstriction andvasopermeation. Am J Respir Cell Mol Biol 2001;24(3):345–51.

33] Lefort J, Singer M, Leduc D, Renesto P, Nahori MA, Huerre M, et al. Systemicadministration of endotoxin induces bronchopulmonary hyperreactivity disso-ciated from TNF-alpha formation and neutrophil sequestration into the murinelungs. J Immunol 1998;161(1):474–80.

34] Leuchte HH, Baezner C, Baumgartner RA, Bevec D, Bacher G, Neurohr C, et al.Inhalation of vasoactive intestinal peptide in pulmonary hypertension. EurRespir J 2008;32(5):1289–94.

35] Linden A, Cardell LO, Yoshihara S, Nadel JA. Bronchodilation by pituitaryadenylate cyclase-activating peptide and related peptides. Eur Respir J1999;14(2):443–51.

36] Liu YC, Khawaja AM, Rogers DF. Effect of vasoactive intestinal peptide (VIP)-related peptides on cholinergic neurogenic and direct mucus secretion in ferrettrachea in vitro. Br J Pharmacol 1999;128(6):1353–9.

37] Lundblad LK, Irvin CG, Adler A, Bates JH. A reevaluation of the validity of unre-strained plethysmography in mice. J Appl Physiol 2002;93(4):1198–207.

38] Matute-Bello G, Frevert CW, Martin TR. Animal models of acute lung injury. AmJ Physiol Lung Cell Mol Physiol 2008;295(3):L379–99.

39] Mirabella N, Squillacioti C, Germano G, Varricchio E, Paino G. Pituitary adenylatecyclase activating peptide (PACAP) immunoreactivity in the ureter of the duck.Cell Tissue Res 2001;305(3):341–9.

40] Moller K, Zhang YZ, Hakanson R, Luts A, Sjolund B, Uddman R, et al. Pituitaryadenylate cyclase activating peptide is a sensory neuropeptide: immuno-cytochemical and immunochemical evidence. Neuroscience 1993;57(3):725–32.

41] Moller M, Fahrenkrug J, Hannibal J. Innervation of the rat pineal gland by pitu-itary adenylate cyclase-activating polypeptide (PACAP)-immunoreactive nervefibres. Cell Tissue Res 1999;296(2):247–57.

42] Mulder H, Uddman R, Moller K, Zhang YZ, Ekblad E, Alumets J, et al. Pitu-itary adenylate cyclase activating polypeptide expression in sensory neurons.Neuroscience 1994;63(1):307–12.

43] Nemeth J, Reglodi D, Pozsgai G, Szabo A, Elekes K, Pinter E, et al. Effect ofpituitary adenylate cyclase activating polypeptide-38 on sensory neuropep-

Journal Identification = PEP Article Identification = 68381 Date: July 8, 2011 Time: 3:18 pm

1 ides 32

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

446 K. Elekes et al. / Pept

tide release and neurogenic inflammation in rats and mice. Neuroscience2006;143(1):223–30.

44] Nemetz N, Abad C, Lawson G, Nobuta H, Chhith S, Duong L, et al. Inductionof colitis and rapid development of colorectal tumors in mice deficient in theneuropeptide PACAP. Int J Cancer 2008;122(8):1803–9.

45] O’Donnell M, Garippa RJ, Rinaldi N, Selig WM, Tocker JE, Tannu SA, et al. Ro 25-1553: a novel, long-acting vasoactive intestinal peptide agonist. Part II: Effecton in vitro and in vivo models of pulmonary anaphylaxis. J Pharmacol Exp Ther1994;270(3):1289–94.

46] Okamoto T, Gohil K, Finkelstein EI, Bove P, Akaike T, van dVA. Multiple con-tributing roles for NOS2 in LPS-induced acute airway inflammation in mice.Am J Physiol Lung Cell Mol Physiol 2004;286(1):L198–209.

47] Onoue S, Ohmori Y, Endo K, Yamada S, Kimura R, Yajima T. Vasoactive intestinalpeptide and pituitary adenylate cyclase-activating polypeptide attenuate thecigarette smoke extract-induced apoptotic death of rat alveolar L2 cells. Eur JBiochem 2004;271(9):1757–67.

48] Pei L. Genomic structure and embryonic expression of the rat type 1vasoactive intestinal polypeptide receptor gene. Regul Pept 1997;71(3):153–61.

49] Rocksen D, Ekstrand-Hammarstrom B, Johansson L, Bucht A, Vitamin E.reduces transendothelial migration of neutrophils and prevents lung injuryin endotoxin-induced airway inflammation. Am J Respir Cell Mol Biol2003;28(2):199–207.

50] Rylander R, Snella MC. Endotoxins and the lung: cellular reactions and risk fordisease. Prog Allergy 1983;33:332–44.

51] Savov JD, Gavett SH, Brass DM, Costa DL, Schwartz DA. Neutrophils play a criticalrole in development of LPS-induced airway disease. Am J Physiol Lung Cell MolPhysiol 2002;283(5):L952–62.

52] Schmidt DT, Ruhlmann E, Waldeck B, Branscheid D, Luts A, Sundler F, et al. Theeffect of the vasoactive intestinal polypeptide agonist Ro 25-1553 on inducedtone in isolated human airways and pulmonary artery. Naunyn SchmiedebergsArch Pharmacol 2001;364(4):314–20.

53] Schoenfeld LK, Souder JA, Hardwick JC. Pituitary adenylate cyclase-activatingpolypeptide innervation of the mudpuppy cardiac ganglion. Brain Res2000;882(1–2):180–90.

54] Skakkebaek M, Hannibal J, Fahrenkrug J. Pituitary adenylate cyclase acti-vating polypeptide (PACAP) in the rat mammary gland. Cell Tissue Res1999;298(1):153–9.

55] Somogyvari-Vigh A, Reglodi D. Pituitary adenylate cyclase activat-

ing polypeptide: a potential neuroprotective peptide. Curr Pharm Des2004;10(23):2861–89.

56] Sreedharan SP, Huang JX, Cheung MC, Goetzl EJ. Structure, expression, and chro-mosomal localization of the type I human vasoactive intestinal peptide receptorgene. Proc Natl Acad Sci U S A 1995;92(7):2939–43.

[

(2011) 1439–1446

57] Strange-Vognsen HH, Arnbjerg J, Hannibal J. Immunocytochemical demon-stration of pituitary adenylate cyclase activating polypeptide (PACAP) in theporcine epiphyseal cartilage canals. Neuropeptides 1997;31(2):137–41.

58] Szakaly P, Laszlo E, Kovacs K, Racz B, Horvath G, Ferencz A, et al. Micedeficient in pituitary adenylate cyclase activating polypeptide (PACAP) showincreased susceptibility to in vivo renal ischemia/reperfusion injury. Neuropep-tides 2011;45(2):113–21.

59] Szema AM, Hamidi SA, Lyubsky S, Dickman KG, Mathew S, Abdel-Razek T,et al. Mice lacking the VIP gene show airway hyperresponsiveness and airwayinflammation, partially reversible by VIP. Am J Physiol Lung Cell Mol Physiol2006;291(5):L880–6.

60] Usdin TB, Bonner TI, Mezey E. Two receptors for vasoactive intestinal polypep-tide with similar specificity and complementary distributions. Endocrinology1994;135(6):2662–80.

61] Vaudry D, Falluel-Morel A, Bourgault S, Basille M, Burel D, Wurtz O, et al. Pitu-itary adenylate cyclase-activating polypeptide and its receptors: 20 years afterthe discovery. Pharmacol Rev 2009;61(3):283–357.

62] Wagner U, Bredenbroker D, Storm B, Tackenberg B, Fehmann HC, von WichertP. Effects of VIP and related peptides on airway mucus secretion from isolatedrat trachea. Peptides 1998;19(2):241–5.

63] Wang ZY, Alm P, Hakanson R. The contribution of nitric oxide to endotoxin-induced ocular inflammation: interaction with sensory nerve fibres. Br JPharmacol 1996;118(6):1537–43.

64] Wang ZY, Alm P, Hakanson R. Distribution and effects of pituitaryadenylate cyclase-activating peptide in the rabbit eye. Neuroscience1995;69(1):297–308.

65] Warren JB, Wilson AJ, Loi RK, Coughlan ML. Opposing roles of cyclic AMP in thevascular control of edema formation. FASEB J 1993;7(14):1394–400.

66] Yoshihara S, Linden A, Kashimoto K, Nagano Y, Ichimura T, Nadel JA. Long lastingsmooth muscle relaxation by a novel PACAP analogue in guinea-pig and primateairways in vitro. Br J Pharmacol 1997;121(8):1730–4.

67] Yoshihara S, Yamada Y, Abe T, Kashimoto K, Linden A, Arisaka O. Long-lastingsmooth-muscle relaxation by a novel PACAP analogue in human bronchi. RegulPept 2004;123(1–3):161–5.

68] Zeldin DC, Wohlford-Lenane C, Chulada P, Bradbury JA, Scarborough PE, RoggliV, et al. Airway inflammation and responsiveness in prostaglandin H synthase-deficient mice exposed to bacterial lipopolysaccharide. Am J Respir Cell MolBiol 2001;25(4):457–65.

69] Zhang YZ, Sjolund B, Moller K, Hakanson R, Sundler F. Pituitary adenylate

cyclase activating peptide produces a marked and long-lasting depression of aC-fibre-evoked flexion reflex. Neuroscience 1993;57(3):733–7.

70] Zhou CJ, Shioda S, Yada T, Inagaki N, Pleasure SJ, Kikuyama S. PACAP and itsreceptors exert pleiotropic effects in the nervous system by activating multiplesignaling pathways. Curr Protein Pept Sci 2002;3(4):423–39.