Role of transient receptor potential vanilloid 1 receptors in endotoxin-induced airway inflammation...

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Role of Transient Receptor Potential Vanilloid 1 receptors in endotoxin-induced airway

inflammation in the mouse

ZSUZSANNA HELYES,*1 KRISZTIÁN ELEKES,1 JÓZSEF NÉMETH,2 GÁBOR

POZSGAI,1 KATALIN SÁNDOR,1 LÁSZLÓ KERESKAI,3 RITA BÖRZSEI,1 ERIKA

PINTÉR,1 ÁRPÁD SZABÓ, 1 AND JÁNOS SZOLCSÁNYI1,2

1Department of Pharmacology and Pharmacotherapy, 2Neuropharmacology Research Group

of the Hungarian Academy of Sciences and 3Department of Pathology,

Faculty of Medicine, University of Pécs, H-7624 Pécs, Szigeti u. 12., Hungary

Zs. Helyes and K. Elekes made equal contributions to the present work.

*Reprint request and correspondence to: Zsuzsanna Helyes, University of Pecs, Faculty of

Medicine, Department of Pharmacology and Pharmacotherapy, H-7624 Pecs, Szigeti u. 12., e-

mail: [email protected], phone: +36-72-536001/5591, fax:+36-72-536218

Running title: Role of TRPV1 in airway inflammation

of 42Articles in PresS. Am J Physiol Lung Cell Mol Physiol (January 19, 2007). doi:10.1152/ajplung.00406.2006

Copyright © 2007 by the American Physiological Society.

mailto:[email protected]

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Abstract

Airways are densely innervated by capsaicin-sensitive sensory neurons expressing Transient

Receptor Potential Vanilloid 1 (TRPV1) receptors/ion channels, which play an important

regulatory role in inflammatory processes via the release of sensory neuropeptides. The aim

of the present study was to investigate the role of TRPV1 receptors in endotoxin-induced

airway inflammation and consequent bronchial hyperreactivity with functional, morphological

and biochemical techniques using receptor gene-deficient mice. Inflammation was evoked by

intranasal administration of E. coli lipopolysaccharide (60 µl, 167 µg/ml) in TRPV1 knockout

(TRPV1-/-) mice and their wild-type counterparts (TRPV1+/+) 24 h before measurement.

Airway reactivity was assessed by unrestrained whole body plethysmography and its

quantitative indicator, enhanced pause (Penh) was calculated after inhalation of the

bronchoconstrictor carbachol. Histological examination and spectrophotometric

myeloperoxidase measurement was performed from the lung. Somatostatin concentration was

measured in the lung and plasma with radioimmunoassay. Bronchial hyperreactivity,

histological lesions (perivascular/peribronchial oedema, neutrophil/macrophage infiltration,

goblet cell hyperplasia) and myeloperoxidase activity were significantly greater in TRPV-/-

mice. Inflammation markedly elevated lung and plasma somatostatin concentrations in

TRPV1+/+, but not in TRPV1-/- animals. In TRPV1-/- mice exogenous administration of

somatostatin-14 (4x100 µg/kg i.p.) diminished inflammation and hyperreactivity.

Furthermore, in wildtype mice antagonizing somatostatin receptors by cyclo-somatostatin

(4x250 µg/kg i.p.) increased these parameters. This study provides the first evidence for a

novel counter-regulatory mechanism during endotoxin-induced airway inflammation, which is

mediated by somatostatin released from sensory nerve terminals in response to activation of

TRPV1 receptors of the lung. It reaches the systemic circulation and inhibits inflammation

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and consequent bronchial hyperreactivity.

Keywords: capsaicin-sensitive afferents; inflammatory airway hyperreactivity;

lipoplysaccharide; myeloperoxidase activity; somatostatin

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The airways are densely innervated by capsaicin-sensitive sensory nerves (54), which play an

important regulatory role in inflammatory processes via the release of sensory neuropeptides.

The Transient Receptor Potential Vanilloid 1 (TRPV1) receptor, also known as capsaicin

receptor, is a non-selective cation channel expressed selectively in the cell membrane of thin

afferent (C and Aδ) fibres, which is activated/sensitized by noxious heat and a variety of

inflammatory mediators, such as protons, lipoxygenase products, bradykinin or prostaglandins

(7; 9; 10; 52). Therefore, its involvement in inflammatory and nociceptive processes has

become an issue with pathophysiological relevance and important scopes for drug

development (7; 52). The gene of the TRPV1 receptor was successfully deleted in mice (10),

which enabled studying the functional roles of this ion channel expressed on these sensory

fibres in animal models of inflammatory diseases.

TRPV1-expressing afferents are not only involved in sensory input, but these nerve

terminals contain several neuropeptides which are released in response to stimulation and

exert effector functions (42; 43). Tachykinins (e.g. substance P: SP, neurokinin A: NKA ) and

calcitonin gene-related peptide (CGRP) elicit neurogenic inflammation and increased

microcirculation, respectively, and induce smooth muscle responses in different visceral

organs (2; 29; 30; 42).

In the respiratory tract SP, NKA and CGRP are also localized in a subpopulation of

afferent fibres and capsaicin evokes pronounced airway inflammation and

bronchoconstriction by releasing these peptides (2; 29; 30; 42). However, more recently a

surprising counter-regulatory humoral function of these nerve endings has been described.

Several lines of evidence indicated that somatostatin released from capsaicin-sensitive

sensory nerve terminals reaches the circulation and elicits systemic anti-inflammatory (16; 41;

44; 45; 50) and analgesic (5) “sensocrine” effects (41). These systemic inhibitory actions of

somatostatin evoked e.g. by antidromic stimulation of the rat sciatic nerve inhibited plasma

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protein extravasation in the trachea and the mediastinal connective tissue (45). Furthermore,

bilateral stimulation of the peripheral stumps of the cut vagal nerves in rats pretreated with

atropine or hexamethonium inhibited neurogenic inflammation in the rat hindpaw skin besides

inducing neurogenic inflammation locally in the trachea (50).

Endotoxins are constituents of the outer layer of gram negative bacteria, they can be

found in the surrounding microenvironment, it is a significant risk factor for asthma and

asthma severity depends to a great extent on its concentration (27; 51). Intranasal

administration of lipopolysaccharide (LPS), the main of component of endotoxins, to mice is

a commonly used confined experimental model to study acute lung inflammation without

causing systemic multiple organ dysfunction (8; 35; 39). Such LPS instillation is known to

induce an acute inflammatory response with a huge influx of neutrophils, consequent

extravasation of plasma proteins into the airways leading to perivascular/peribronchial edema

formation (8; 39) and activation of macrophages (27).

The aim of the present study was to analyse the modulatory role of the TRPV1

capsaicin receptor/ion channel in endotoxin-induced airway inflammation and consequent

bronchial hyperreactivity using TRPV1 gene-deleted mice and participation of the anti-

inflammatory sensory neuropeptide somatostatin in these responses.

METHODS

Animals. Experiments were performed on female TRPV1 receptor gene knockout mice

(TRPV1-/-) and their wild-type counterparts (TRPV1+/+) weighing 20-25 g. Two breeding pairs

of TRPV1+/- heterozygote mice were given as generous gifts from Dr. J.B. Davis

(GlaxoSmithKline, Harlow, U.K.). The genotype of the animals in the first generation was

determined by Southern blot analysis and PCR. The TRPV1+/+ and TRPV1-/- were successfully

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bred on as wild-type and knockout mice in the Laboratory Animal Centre of the University of

Pécs under standard pathogen free conditions at 24-25 °C provided with standard chow and

water ad libitum, from where they were obtained for the experiments.

Both experimental series presented in this paper were performed in blocks with 10-12 mice

per day. Each group presented in one figure ran at the same time. Animals were randomised

to the treatments and mice belonging to every group were assessed for Penh simultaneously

every day.

Generation of TRPV1 receptor knockout mice. The generation of TRPV1 receptor

knockout mice was by homologous recombination in embryonic stem cells (129 ES) to

generate a mouse lacking transmembrane domains 2-4 of the mTRPV1 gene. Germline

chimaeras were crossed onto C57BL/6 females to generate heterozygotes, which were inter-

crossed giving rise to healthy homozygous mutant offsprings in the expected Mendelian ratio,

as described by Davis and co-workers (10). Succesful targeting of the locus and germline

transmission was confirmed by polymerase chain reaction (PCR) and Southern blot analysis.

After genotyping by PCR, they were bred from homozygous knockout breeding pairs, so all

offsprings were also homozygous knockouts (TRPV1-/-), as described.

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Induction of airway inflammation. Subacute airway inflammation was evoked by 60 µl

Escherichia coli (serotype: 083) LPS (167 µg/ml dissolved in sterile PBS; Sigma, St. Louis,

MO, USA) applied intranasally 24 h prior to measurement (35). Data showing that intranasal

administration of this LPS dose evoked maximal inflammation (neutrophil accumulation and

inflammatory cytokine production) 24 h after its instillation served as basis for chosing this

time point. The same volume of sterile PBS was administered to control mice regarded as

intact (uninflamed) animals.

Determination of airway responsiveness. Airway responsiveness in conscious,

spontaneously breathing animals was measured by recording respiratory pressure curves by

whole body plethysmography (Buxco Europe Ltd, Winchester, UK) (11; 31). Aerosolized

saline and then the muscarinic acetylcholine receptor agonist carbachol (carbamoyl-β-

methylcholine; Sigma, St. Louis, MO, USA) in increasing concentrations (50 µl per mouse

for 1.5 min in 5.5, 11, 22 and 44 mM concentrations) were nebulized through an inlet of the

main chamber for 50 sec to induce bronchoconstriction 24 h after LPS administration and

readings were taken and averaged for 15 minutes following each nebulization. Baseline values

usually returned at the end of this period. Enhanced pause (Penh) was measured as an

indicator of bronchoconstriction and consequent increase of airway resistance. Penh is a

complex, calculated parameter ((expiratory time/ relaxation time)-1): (max. expiratory flow/

max. inspiratory flow), which closely correlates with airway resistance as measured by

traditional invasive techniques using ventilated animals (11; 32; 33). Percentage increase of

the enhanced pause (Penh) above baseline ((penh in response to the respective carbachol

concentration- baseline penh/baseline penh) x 100) was calculated in each 15 min period after

respective carbachol stimulations.

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For examining if Penh correlates with lung resistance (Rl: ∆cmH2O * sec/ml) in the

present model, direct measurement of Rl was also performed in tracheotomized and

mechanically ventilated mice. Animals were anaesthetized with ketamine and xylazine, a

stainless 18G tube was inserted into the trachea and tied into place. The tracheostomy cannula

was passed through a hole in the whole body plethysmograph (Buxco Europe Ltd,

Winchester, UK). A connector was attached to this tube with two ports connected to the

inspiratory and expiratory sides of a ventilator (MiniVent Type 845, Hugo Sachs Electronik,

Harvard Apparatus, Germany). Ventilation was achieved at 150 breaths per minute and a VT

of 0.2 ml with a positive end-expiratory pressure of 2-4 cm H2O. The flow was measured by

digital differentiation of the volume signal and the Rl was continuously computed by fitting

flow, volume and pressure to an equation of motion using a recessive least squares algorithm

(47). Aerosolized carbachol was administered through bypass tubing via the ultrasonic

nebulizer following the same protocol as described for the unrestrained measurement. The

percentage changes of Rl above baseline induced by each carbachol concentration was

calculated and compared to the percentage changes of Penh.

At the end of the measurement mice were anaesthesized with ketamine (100 mg/kg

i.p.; Calypsol; Richter-Gedeon Ltd., Budapest, Hungary) and xylazine (10 mg/kg i.m.;

Xylavet; Phylaxia-Sanofi, Veterinary Biology Co. Ltd., Budapest, Hungary), blood samples

were taken by cardiac puncture, then killed by cervical dislocation and their lungs were

excised.

Histological studies and scoring. Lung specimens were fixed in 4% formaldehyde for 8 h,

embedded in paraffin, sectioned with microtome (5-7 µm) and stained with hematoxylin

(Sigma, St. Louis, MO, USA) and eosin (Molar Chemicals Ltd., Budapest, Hungary) or

periodic acid-Schiff (PAS; Sigma, St. Louis, MO, USA) to more precisely visualize mucus

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producing goblet cells. Semiquantitative scoring of the inflammatory changes was performed

by an expert pathologist blinded from the study as described by Zeldin et al (55) on the basis

of the presence or abundance of the following: (1) perivascular oedema (0:absent; 1: mild to

moderate, involving fewer than 25% of the perivascular spaces; 2: moderate to severe,

involving more than 25% but less than 75% of perivascular spaces; 3: severe involving more

than 75% of perivascular spaces); (2) perivascular/ peribronchial acute inflammation (0:

absent; 1: mild acute inflammation in the perivascular oedematous space with fewer than 5

neutrophils per high-power field; 2: moderate acute inflammation in the perivascular spaces

extending to involve the peribronchial spaces with more than 5 neutrophils per hpf in these

regions; 3: severe acute inflammation in the perivascular and peribronchial spaces with

numerous neutrophils encircling most bronchioles; (3) goblet cell metaplasia of the

bronchioles (0: absent; 1: few goblet cells present in one or two bronchioles; 2: large number

of goblet cells present); (4) macrophages/ mononuclear cells in the alveolar spaces (0: absent;

1: present in fewer than 25% of alveolar spaces, 2: >25% of alveolar spaces). The score

values for these individual parameters were added to form a composite inflammatory score

(ranging from 0 to 10). From every specimen (8-10 mice in each group) 4-5 sections were

taken from different depths to give a representative appreciation of the whole lung. Mean

scores were determined from the different sections of the individual animals and composite

score values of the different experimental groups were calculated from these mean scores.

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

granulocytes, especially neutrophils, was determined from the frozen lung samples by

assessment of MPO activity. Lung pieces were thawed and chopped into small pieces then

homogenized in 4 ml 20 mM potassium-phosphate buffer (pH 7.4). The homogenate was

centrifuged at 10,000g at 4 ºC for 10 min and supernatant was removed for somatostatin

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radioimmunoassay (see below). The pellet was resuspended in 4 ml 50 mM potassium-

phosphate buffer containing 0.5% hexadecyl-trimethyl-ammonium-bromide (pH 6.0) and

centrifuged again. Neutrophil accumulation was assessed by comparing MPO enzyme activity

of the samples to a human standard MPO preparation (Sigma, St. Louis, MO, USA). MPO

activity was assayed from the supernatant using H2O2-3,3’,5,5`-tetramethyl-benzidine

(TMB/H2O2) (Sigma, St. Louis, MO, USA). Reactions were performed in 96-well microtitre

plates in room temperature. The optical density (OD) at 620 nm was measured at 5 min

intervals for 30 min, using a microplate reader (Labsystems) and plotted. The reaction rate (∆

OD/time) was derived from an initial slope of the curve. A calibration curve was then

produced, with the rate of reaction plotted against the standard samples (1).

Measurement plasma and lung somatostatin concentrations Concentration of

somatostatin from the plasma and supernatants obtained from lung homogenates was

determined with a specific and sensitive radioimmunoassay (RIA) technique developed in our

laboratory (16; 34). The animals were fasted overnight before the measurement to avoid

gastrointestinal somatostatin release. Arterial blood samples (0.8-1 ml per animal) were taken

into ice-cold Eppendorf tubes containing EDTA (2 mg) and Trasylol (350 U) at the end of the

experiment, 25 h after LPS administration. Following centrifugation (2200 rpm for 10 min at

4°C) the peptide was extracted from the plasma by addition of 3 volumes of absolute alcohol.

After precipitation and a second centrifugation (2000 rpm for 10 min at 4°C) the samples

were dried under nitrogen flow and then resuspended in assay buffer before RIA

determination (34).

Drug treatments. Somatostatin-14 (SOM-14; 100 µg/kg i.p.; Sigma, St. Louis, MO, USA)

was administered to study its actions on the inflammatory response both in TRPV1-/- and

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TRPV1+/+ animals. For examining the functional roles of somatostatin released via TRPV1

activation in LPS-induced airway inflammation, a separate group of TRPV1+/+ mice was

treated with the somatostatin receptor antagonist cyclo-somatostatin (C-SOM; 250 µg/kg i.p.;

Sigma, St. Louis, MO, USA). Both somatostatin and cyclo-somatostatin were dissolved

freshly in saline and injected 30 min before the administration of LPS and every 6 h during

the 24 h experimental period. Mice in the control groups received the same volume of saline.

There were 6-12 animals in each experimental group.

Statistical analysis. Values for all measurements were expressed as the mean + SEM of

n=8-14 mice in each group. Penh data were expressed as % increase above baseline ((penh in

response to the respective carbachol concentration- baseline penh/baseline penh) x 100). AUC

for the carbachol concentration-response curves were calculated and compared with two way

ANOVA. Histological inflammatory score values were analysed with Kruskal-Wallis

followed by Dunn’s test. Data for plasma and lung somatostatin concentrations and lung MPO

activity were evaluated by two-way ANOVA followed by Bonferroni’s modified t-test. In all

cases p<0.05 was considered to be significant.

Ethics All experimental procedures were carried out according to the 1998/XXVIII Act of the

Hungarian Parliament on Animal Protection and Consideration Decree of Scientific

Procedures of Animal Experiments (243/1988) and complied with the recommendations of

the Helsinki Declaration. The studies were approved by the Ethics Committee on Animal

Research of Pécs University according to the Ethical Codex of Animal Experiments and

licence was given (licence No.: BA 02/200-6-2001).

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RESULTS

Inflammatory airway hyperresponsiveness in TRPV1+/+ and TRPV1-/- mice.

Baseline Penh and basal lung resistance (Rl) significantly increased after intranasal LPS

instillation compared to untreated mice both in the TRPV1+/+ (2.42+0.43 vs 0.85+0.08 and

3.23+0.45 vs. 0.69+0.06, respectively) and the TRPV1-/- (2.41+0.67 vs 0.95+0.15 and

2.69+0.23 vs. 0.86+0.09, respectively) groups. Inhalation of the muscarinic receptor agonist

carbachol evoked a concentration-dependent bronchoconstriction shown by the Penh curves,

as well as the Rl values. Responses demonstrated as percentage increase of Penh and Rl above

baseline were markedly enhanced in the LPS-treated groups compared to the respective non-

inflamed controls pointing out the development of inflammatory bronchial

hyperresponsiveness. These results provided evidence that the changes of Penh well correlate

with the changes of Rl in the present experimental model (Table 1.).

There was no significant difference between the carbachol-induced responses of intact

(uninflamed) TRPV1+/+ and TRPV1-/- animals (p=0.75). In response to LPS-induced

inflammation both maximal Penh values were higher and the duration of the

bronchoconstriction was prolonged in mice lacking the TRPV1 receptor. Therefore, the

percentage increase of the mean Penh values evoked by 11, 22 and 44 mM carbachol

inhalation was markedly greater in TRPV1 -/- mice than in their wildtype counterparts (Table

1., Fig. 1.). Area under the carbachol concentration-response curves were 104.7+9.2 and

179.9+11.4 units in the intact TRPV1+/+ and TRPV1-/- groups, respectively. In comparison,

the corresponding data in LPS-treated mice were 503.9+4.6 (p=0.0074 vs. intact TRPV1+/+

mice) and 1211.0+19.8 units (p=0.0002 vs. intact TRPV1-/- mice and p=0.0085 vs. LPS-

treated TRPV1+/+ mice), respectively.

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Inflammatory changes in the lung of TRPV1+/+ and TRPV1-/- mice. Histological

examination and scoring revealed that compared to the intact lung structure (Fig. 2A.), LPS

induced marked peribronchial/ perivascular oedema formation, granulocyte accumulation

around the bronchi, infiltration of mononuclear cells and hyperplasia of mucus producing

goblet cells. All these inflammatory parameters were significantly more severe in TRPV1

receptor gene-deficient mice than in their wildtype counterparts (p=0.0069 intact vs. LPS-

treated TRPV1+/+ mice; p=0.000078 intact vs. LPS-treated TRPV1-/- mice; p=0.019 LPS-

treated TRPV1+/+ vs. LPS-treated TRPV1-/-) (Fig. 2.).

Myeloperoxidase activity in the lung of TRPV1+/+ and TRPV1-/- mice. Endotoxin

administration induced about 2-fold and 4-fold elevations of MPO activity in the lung of

TRPV1+/+ (p=0.005) and TRPV1-/- mice (p=0.0002), respectively. This quantitative marker of

accumulated granulocytes in the inflamed tissue was significantly greater, more than double

in the TRPV1 receptor knockout group (p=007) (Fig. 3.).

Somatostatin concentration in the lung and plasma of TRPV1+/+ and TRPV1-/- mice. The

basal level of somatostatin-like immunoreactivity was about 4-5 folds higher in the lung than

in the plasma of both wildtype and TRPV1 gene-deleted animals without a significant

difference between the two groups (p=0.75). In response to LPS administration there was a

pronounced increase of somatostatin-like immunoreactivity in the lung and plasma of

TRPV1+/+ mice (p=0.02 and p=0.0031), while in the TRPV1-/- group the LPS-induced

elevation of somatostatin level was much smaller in the lung (p=0.16 intact vs. LPS-treated;

p=0.028 LPS-treated TRPV1+/+ vs. TRPV1-/-) and absent in the plasma (p=0.92; p=0.002

LPS-treated TRPV1+/+ vs. TRPV1-/-). These results point out a TRPV1 receptor-mediated

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release of this neuropeptide from the sensory fibres of the lung, which gets into the systemic

circulation (Fig. 4.).

Role of somatostatin in endotoxin-evoked airway hyperresponsiveness, inflammatory

histopathological changes and myeloperoxidase activity. Repeated treatments of TRPV1-/-

with somatostatin-14 (100 µg/kg i.p.) markedly inhibited bronchoconstriction induced by 11,

22 and 44 mM carbachol with the maximal effect reached at 22 mM concentration. Similarly,

the same doses of somatostatin also significantly diminished inflammatory airway

hyperreactivity in TRPV1+/+ mice, although the degree of the inhibitory effect was smaller.

Meanwhile, cyclo-somatostatin (250 µg/kg i.p.), which antagonizes the effects of somatostatin

at all the 5 receptor subtypes, significantly enhanced bronchial hyperreactivity in TRPV1+/+

mice. In the LPS-treated TRPV1+/+ group AUC for the carbachol concentration-response

curves was 503.9+4.6 units, which was significantly smaller after repeated somatostatin-14

injections (323.5+2.6; p=0.012) and greater following cyclo-somatostatin administrations

(1676.5+12.4; p=0.0062). The corresponding AUC values in LPS-treated TRPV1-/- mice was

1211.0+19.8 units (p=0.0085 vs. LPS-treated TRPV1+/+ mice) and it was also markedly

decreased by somatostatin injections (370.8+3.4; p=0.0014) (Fig. 5.).

Somatostatin administration diminished endotoxin-induced inflammatory changes both in

knockout mice (p=0.02) and their wildtype counterparts (p=0.04). Cyclo-somatostatin

injection in the TRPV1+/+ group aggravated these parameters, especially peribronchial

oedema formation, granulocyte infiltration and goblet cell hyperplasia (p=0.027) (Figs. 6., 7.).

In accordance with these histological findings somatostatin significantly decreased LPS-

evoked MPO activity in the lung of TRPV1-/- mice (p=0.007), as well as in TRPV1+/+ animals

(p=0.037). Administration of the antagonist induced a more than 2-fold elevation in the

TRPV1+/+ group compared to their controls (p=0.0082) (Fig. 8.).

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Administration of cyclo-somatostatin (250 µg/kg i.p., 4 times) to TRPV1-/- mice in LPS-

induced inflammation did not alter bronchial hyperreactivity and the inflammatory changes.

No change was observed in carbachol-induced bronchoconstriction, histological parameters

and MPO activity after repeated administration of the same doses of the antagonist, as well as

somatostatin-14 (100 µg/kg i.p., 4 times) to intact TRPV1+/+ and TRPV1-/- mice as compared

to the respective intact animals.

DISCUSSION

The present results demonstrate that endotoxin-induced airway inflammation as

revealed by myeloperoxidase activity measurement and histological assessments, as well as

consequent bronchial hyperreactivity are enhanced in TRPV1 gene-deleted mice compared to

their wildtype counterparts. Activation of the TRPV1 capsaicin receptor in different tissues

elicits neurogenic inflammation and nociception, which are absent in TRPV1-/- animals (6).

On the contrary, inflammatory and pain responses evoked by several other agents are

differently influenced by the TRPV1 receptor. In TRPV1-null mutant mice neurogenic

inflammation induced by mustard oil (1), oedema evoked by carrageenin or mechanical

hyperalgesia one day after the injection of complete Freund’s adjuvant into the hindpaw were

similar to the TRPV1+/+ controls (6). Meanwhile, thermal hyperalgesia in these acute

inflammatory models (10) and adjuvant-induced chronic arthritis and consequent mechanical

hyperalgesia (40) were markedly decreased in TRPV1 receptor knockout mice. On the

contrary, this receptor proved to have a protective role against the development of mechanical

allodynia under diabetic and toxic polyneuropathy conditions (5). Based on these data,

TRPV1 can be considered as an integrator target molecule for a variety of noxious stimuli and

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inflammatory mediators (21; 22; 52) and its function seems to depend on the pathomechanism

of the disease.

In the present series of experiments lung function in unrestrained mice was assessed

by whole body plethysmography and bronchoconstriction was determined on the basis of

Penh. Although this calculated parameter as an appropriate indicator of bronchoconstriction

has been questioned by some authors (31), several data in literature suggest that it is

acceptable for the evaluation of airway reactivity (11; 14; 15). The reason for this

contradiction might be differences in species, experimental models and severity/type of the

inflammation. Our results provided evidence that in the presently used LPS-induced lung

inflammation model there is a relatively good correlation between carbachol-induced Penh

responses and lung resistance measured in tracheotomized, mechanically ventilated animals,

which is supported by other authors using this parameters to determine airway reactivity (11;

14; 15; 39).

Endotoxin (LPS), a constituent of Gram-negative bacteria found in the environment

including house dust (51), induces powerful inflammatory effects on the murine airways

accompanied by bronchopulmonary hyperreactivity, increased responsiveness to

bronchoconstrictor agents, e.g. muscarinic receptor agonists (28). Intranasal endotoxin

administration in the present experimental model evokes a well-defined acute airway

inflammation involving infiltration of granulocytes and cytokine, mainly TNF-α and IL-6,

production (38; 39). Neutrophil activation, as opposed to recruitment alone, probably

accounts for LPS-induced inflammation and consequent bronchial hyperreactivity, since their

depletion is fully suppressive (39). They are recruited to the airway epithelial/ subepithelial

regions and cause tissue damage via the production and release of oxygen radicals, proteases,

cytokines and chemokines (25), which attract and stimulate mononuclear cells and

lymphocytes. All these inflammatory and immune cells release several other inflammatory

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mediators like leukotrienes, prostaglandins, bradykinin etc., which can activate a variety of

receptors including TRPV1 on the sensory nerves in the airways (3; 29). The released

neuropeptides in turn influence the inflammatory process by acting at receptors localized on

these peripheral nerve terminals themselves, vascular endothelial, bronchial epithelial and

inflammatory cells (53). There is increasing evidence that neuropeptides are synthesized in

non-neural sources as well, e.g. mononuclear cells and lymphocytes, and they can be released

in a TRPV1-independent manner, especially under inflammatory conditions. Pro-

inflammatory neuropeptides like substance P, neurokinin A and CGRP are localized in

capsaicin-sensitive unmyelinated and thinly myelinated sensory fibres (C- and Aδ fibres) in

the airways of several species (24), their release from these nerve terminals and participation

in the enhancement of both vascular and cellular phases of several inflammatory reactions is

well established (4; 29; 53). Besides these pro-inflammatory neuropeptides, somatostatin is

also present in the capsaicin-sensitive afferents of the lung (19). However,

immunohistochemical studies have provided evidence that somatostatin and substance P/

CGRP are localized in distinct subpopulations of primary sensory neurons (19; 32).

This study revealed that intranasal endotoxin administration increased somatostatin

concentration in the lung and also in the plasma of wildtype mice. In the TRPV1 gene-deleted

group LPS-induced pronounced rise of somatostatin concentration was abolished in the

plasma and markedly inhibited in the lung. These results indicate that inflammatory mediators

stimulate the TRPV1 receptor on sensory nerve terminals and elicit the release of somatostatin

from these afferents. The increase of plasma somatostatin level was similar to that observed in

response to topical application of mustard oil on the rat hindpaw skin, which was also

sufficient to evoke systemic anti-inflammatory actions (46). In accordance with these findings

in TRPV1 knockout mice enhanced inflammatory reaction was observed in the lung in

response to endotoxin administration. This is likely to be explained by LPS-induced

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production of an “inflammatory mixture” containing lipoxygenase products, protons,

prostaglandins, bradykinin, etc., which all activate/sensitize the integrative TRPV1 ion

channel localized on somatostatin-containing capsaicin-sensitive fibres in the airways (40;

44), although even in this model some involvement of decreased tachykinin release cannot be

ruled out in TRPV1-/- mice.

In LPS-treated TRPV1-/- mice exogenous administration of somatostatin-14 prevented

both increased bronchoconstriction and enhanced inflammatiory reaction determined by MPO

and histology. On the other hand, inhibitory function of the released somatostatin in the

development airway inflammation and hyperresponsiveness was further evidenced in

wildtype mice treated with the somatostatin receptor antagonist cyclo-somatostatin. This

compound which inhibits the actions of somatostatin at all the five receptor subtypes (sst1-

sst5) (20; 36) enhanced both inflammatory responses and bronchial hyperreactivity.

Somatostatin effectively inhibits the release of pro-inflammatory neuropeptides (36;

37) and modulates the immune system by inhibiting monocyte-macrophage functions (26), B

lymphocyte immunoglobulin production, T lymphocyte proliferation and cytokine production

(23; 36). All the 5 different receptor subtypes (sstr1-5) belong to the G protein-coupled

receptor family (20). In the lung they have been detected on lymphocytes and also on the

sensory nerve terminals (13). Somatostatin receptor autoradiography has shown upregulation

of somatostatin binding sites in several immune-mediated diseases (36; 48; 49). Furthermore,

plasma somatostatin level has been shown to significantly increase during the development of

adjuvant-induced arthritis in the rat (16) and according to the present results also during

endotoxin-induced airway inflammation in the mouse.

Our data obtained in the same LPS-induced airway inflammation model suggest that

tachykinins (e.g. substance P) and CGRP participate in neutrophil accumulation and in the

production of the inflammatory cytokine interleukin-1β, but do not affect the overall severity

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of this type of inflammatory reaction (12). However, in the present model these inflammatory

actions of the released pro-inflammatory sensory neuropeptides are counteracted by the anti-

inflammatory somatostatin also derived from the C-fibres in response to TRPV1 receptor

activation. Since somatostatin and its synthetic agonists have previously been proved to

inhibit the outflow of these neuropeptides from isolated rat tracheae (17; 18), the ability of

somatostatin released from sensory nerves to diminish the release of substance P and CGRP

might be involved in its inhibitory effect.

These data provide the first evidence for a TRPV1 receptor-dependent novel type of

counter-regulatory mechanism developing during airway inflammation, since endotoxin-

induced inflammation and consequent bronchial hyperresponsiveness were enhanced in

TRPV1 gene-deficient mice. In this model somatostatin released from sensory fibres of the

lung via TRPV1 receptor stimulation, mediates these inhibitory actions at the site of

activation and also reaches the systemic circulation. The somatostatin receptor antagonist

cyclo-somatostatin prevents this counter-regulatory effect and aggravates endotoxin-induced

responses indicating that synthetic somatostatin receptor agonists might be potential novel

agents for the treatment of airway inflammation.

Acknowledgements: This work was supported by Hungarian Grants OTKA F-046635, T-049027,

T-046729, T-043467, RET-008/2005, ETT-284/2006. Buxco Whole Body Plethysmograph was

purchased with financial support of GVOP-3.2.1-2004-04-0420/3.0. We acknowledge Dr. J.B. Davis,

GlaxoSmithKline, R&D Ltd. U.K. for the generous gift of TRPV1 knockout mice. The authors thank

Mrs. Anikó Perkecz for the histological slides.

Grants: Hungarian Grants OTKA F-046635, T-049027, T-046729, T-043467, ETT-

284/2006, RET-008/2005 and NRDP1A/005/2004. Buxco Whole Body Plethysmograph was

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purchased with financial support of GVOP-3.2.1-2004-04-0420/3.0. We acknowledge Dr. J.B.

Davis, GlaxoSmithKline, R&D Ltd. U.K. for the generous gift of TRPV1 knockout mice.

Conflict of Interest Statement: None of the authors has a financial relationship with a

commercial entity that has an interest in the subject of this manuscript.

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Figure Legends

Fig. 1. Endotoxin-induced bronchial hyperreactivity to inhaled carbachol

Bronchoconstriction induced by increasing concentrations of carbachol was assessed in freely

moving, unrestrained mice by whole body plethysmography. Percentage increase of the

enhanced pause (Penh) above baseline ((penh in response to the respective carbachol

concentration- baseline penh/baseline penh) x 100) was calculated in each 15 min period after

respective carbachol stimulations. Data points represent means+S.E.M. of n= 8-14

experiments. Area under these carbachol concentration-response curves (AUC) were

calculated and compared with two-way ANOVA (p=0.0074 LPS-treated TRPV1+/+ mice vs.

respective intact group; p=0.0002 LPS-treated TRPV1-/- mice vs. respective intact group and

p=0.0085 LPS-treated TRPV1+/+ vs. LPS-treated TRPV1-/- mice).

Fig. 2. Histopathological examination of lung samples

LPS-induced inflammatory histopathological changes in lung samples of TRPV1+/+ wildtype

(B) and TRPV1-/- mice (C) compared to the structure of the intact lung (A). Samples are

stained with hematoxylin and eosin and shown at 200x magnification; a: alveolar spaces, b:

bronchi, v: vessels. Panel 2D. Semiquantitative evaluation and scoring of the lung samples on

the basis of perivascular oedema formation, perivascular/peribronchial granulocyte

accumulation, goblet cell hyperplasia and alveolar mononuclear cell infiltration. Each column

represents the means+S.E.M. of n= 8-14 mice; +p<0.01 LPS-treated inflamed vs. respective

intact mice; *p<0.05 LPS-treated TRPV1-/- vs. LPS-treated TRPV1+/+ group (Kruskal-Wallis

followed by Dunn’s test).

Fig. 3. Myeloperoxidase (MPO) activity of lung samples

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Myeloperoxidase activity, as a quantitative indicator of the number of accumulated

granulocytes determined from homogenized lung samples of TRPV1+/+ and TRPV1-/- mice

25h after intranasal administration of LPS compared to the respective intact animals. Results

are means+S.E.M. of n= 8-14 mice; *p<0.01 LPS-treated inflamed vs. respective intact mice

and +p<0.01 TRPV1-/- vs. TRPV1+/+ group (two-way ANOVA followed by Bonferroni’s

modified t-test).

Fig. 4. Somatostatin concentrations

Concentrations of somatostatin measured with radioimmunoassay in the lung (A) and plasma

(B) of TRPV1+/+ and TRPV1-/- mice. Columns show mean results+S.E.M. of n= 8-14

mice;*p<0.05, **p<0.01 LPS-treated inflamed vs. the respective intact group and +p<0.05,

++p<0.01 TRPV1-/- vs. TRPV1+/+ mice (two-way ANOVA followed by Bonferroni’s modified

t-test).

Fig. 5. Role of somatostatin in endotoxin-induced bronchial hyperreactivity

Effect of somatostatin-14 (SOM-14) and the somatostatin receptor antagonist cyclo-

somatostatin (C-SOM) on LPS-induced inflammatory bronchial hyperreactivity in TRPV1-/-

and TRPV1+/+ mice, respectively. Bronchoconstriction induced by increasing concentrations

of carbachol was assessed in freely moving, unrestrained mice by whole body

plethysmography. Percentage increase of the enhanced pause (Penh) above baseline ((penh in

response to the respective carbachol concentration- baseline penh/baseline penh) x 100) was

calculated in each 15 min period after respective carbachol stimulations. Data points represent

means+S.E.M. of n= 8-14 experiments. Area under the carbachol concentration-response

curves (AUC) were calculated and compared with two way ANOVA (p=0.0085 LPS-treated

TRPV1+/+ vs. LPS-treated TRPV1-/- mice; p=0.012 TRPV1+/+, LPS vs. TRPV1+/+, LPS+SOM-

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14; p=0.0062 TRPV1+/+, LPS vs. TRPV1+/+, LPS+C-SOM and p=0.0014 TRPV1-/-, LPS vs.

TRPV1-/-, LPS+SOM-14).

Fig. 6. Role of somatostatin in endotoxin-induced inflammatory histological changes

LPS-induced inflammatory changes in lung samples obtained from TRPV1+/+ (A), TRPV1-/-

(B), cyclo-somatostatin treated TRPV1+/+ (C) and somatostatin-14-treated TRPV1-/- (D) mice.

Staining was performed with periodic acid-Schiff (PAS) for better identification of mucus

producing bronchial goblet cells and shown at 200x magnification; a: alveolar spaces, b:

bronchi, v: vessels.

Fig. 7. Composite histopathological scores demonstrating the role of somatostatin in

endotoxin-induced inflammation

Semiquantitative evaluation and scoring of the lung samples on the basis of perivascular

oedema formation, perivascular/peribronchial granulocyte accumulation, goblet cell

hyperplasia and alveolar mononuclear cell infiltration. Each column represents the

means+S.E.M. of n= 8-14 mice; *p<0.05 TRPV1-/- vs. TRPV1+/+ group; #p<0.05 cyclo-

somatostatin (C-SOM)-treated vs. TRPV1+/+; +p<0.05 somatostatin-14 (SOM-14)-treated vs.

respective control group (Kruskal-Wallis followed by Dunn’s test).

Fig. 8. Role of somatostatin in endotoxin-induced granulocyte accumulation in the lung

Myeloperoxidase (MPO) activity, as a quantitative indicator of the number of accumulated

granulocytes, determined from homogenized lung samples 25h after intranasal administration

of LPS. The effect of cyclo-somatostatin (C-SOM) treatment in TRPV1+/+ mice and

somatostatin-14 (SOM-14) administration in both TRPV1+/+ and TRPV1-/- animals are shown.

Results are means+S.E.M. of n= 8-14 mice; *p<0.01 TRPV1-/- vs. TRPV1+/+ group; #p<0.01

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cyclo-somatostatin (C-SOM)-treated vs. TRPV1+/+; +p<0.05; ++p<0.01 somatostatin-14

(SOM-14)-treated vs. respective control group (Kruskal-Wallis followed by Dunn’s test).

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Concentration ofcarbachol

(mM)

Parameters

5.5 11 22

% changes of penh TRPV+/+

Intact 2.7±0.7 4.6±4.1 58 .9±1.0 LPS-treated 56.1±12.1 99.6±29.4 191.1±33.2

TRPV-/- Intact 24.1±5.4 33.8±13.5 71.0±6.5

LPS-treated 88.3±20.5 280.0±88.7 527.9±125.2 % changes of Rl

TRPV+/+ Intact 3.8±0.9 6.8±1.3 71.7±21.8

LPS-treated 48.4±3.1 103.3±23.3 210.8±45.5 TRPV-/-

Intact 27.2±13.7 52.9±17.0 94.2±30.6 LPS-treated 96.1±14.3 263.6±67.7 574.4±99.9

Table 1. Percentage increase of mean enhanced pause (Penh) and mean lung resistance (Rl; ∆cmH2O*sec/ml) values from baseline in response to increasing concentrations of carbachol. Data are normalized to the respective baseline values: [(responses for each carbachol concentration – respective baseline responses) / baseline response] x 100 and presented as means + S.E.M. of n= 8-14 experiments. Enhanced pause (Penh) was determined with unrestrained whole body plethysmography in conscious mice and lung resistance was measured directly in tracheotomized, mechanically ventilated animals to compare these two ways of airway function measurements and to prove that Penh well correlates with bronchoconstriction in the present endotoxin-induced inflammation model.

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Fig. 1. Endotoxin-induced bronchial hyperreactivity to inhaled carbachol Bronchoconstriction induced by increasing concentrations of carbachol was assessed in freely moving, unrestrained mice by whole body plethysmography. Percentage increase

of the enhanced pause (Penh) above baseline ((penh in response to the respective carbachol concentration- baseline penh/baseline penh) x 100) was calculated in each 15 min period after respective carbachol stimulations. Data points represent means+S.E.M.

of n= 8-14 experiments. Area under these carbachol concentration-response curves (AUC) were calculated and compared with two-way ANOVA (p=0.0074 LPS-treated

TRPV1+/+ mice vs. respective intact group; p=0.0002 LPS-treated TRPV1-/- mice vs. respective intact group and p=0.0085 LPS-treated TRPV1+/+ vs. LPS-treated TRPV1-/-

mice).

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Fig. 2. Histopathological examination of lung samples LPS-induced inflammatory histopathological changes in lung samples of TRPV1+/+ wildtype (B) and TRPV1-/- mice

(C) compared to the structure of the intact lung (A). Samples are stained with hematoxylin and eosin and shown at 200x magnification; a: alveolar spaces, b: bronchi, v:

vessels. Panel 2D. Semiquantitative evaluation and scoring of the lung samples on the basis of perivascular oedema formation, perivascular/peribronchial granulocyte

accumulation, goblet cell hyperplasia and alveolar mononuclear cell infiltration. Each column represents the means+S.E.M. of n= 8-14 mice; +p<0.01 LPS-treated inflamed vs. respective intact mice; *p<0.05 LPS-treated TRPV1-/- vs. LPS-treated TRPV1+/+ group

(Kruskal-Wallis followed by Dunn's test).

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Fig. 3. Myeloperoxidase (MPO) activity of lung samples Myeloperoxidase activity, as a quantitative indicator of the number of accumulated granulocytes determined from homogenized lung samples of TRPV1+/+ and TRPV1-/- mice 25h after intranasal

administration of LPS compared to the respective intact animals. Results are means+S.E.M. of n= 8-14 mice; *p<0.01 LPS-treated inflamed vs. respective intact mice and +p<0.01 TRPV1-/- vs. TRPV1+/+ group (two-way ANOVA followed by Bonferroni's

modified t-test).

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Fig. 4. Somatostatin concentrations Concentrations of somatostatin measured with radioimmunoassay in the lung (A) and plasma (B) of TRPV1+/+ and TRPV1-/- mice. Columns show mean results+S.E.M. of n= 8-14 mice;*p<0.05, **p<0.01 LPS-treated

inflamed vs. the respective intact group and +p<0.05, ++p<0.01 TRPV1-/- vs. TRPV1+/+ mice (two-way ANOVA followed by Bonferroni's modified t-test).

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Fig. 5. Role of somatostatin in endotoxin-induced bronchial hyperreactivity Effect of somatostatin-14 (SOM-14) and the somatostatin receptor antagonist cyclo-somatostatin

(C-SOM) on LPS-induced inflammatory bronchial hyperreactivity in TRPV1-/- and TRPV1+/+ mice, respectively. Bronchoconstriction induced by increasing concentrations

of carbachol was assessed in freely moving, unrestrained mice by whole body plethysmography. Percentage increase of the enhanced pause (Penh) above baseline ((penh in response to the respective carbachol concentration- baseline penh/baseline

penh) x 100) was calculated in each 15 min period after respective carbachol stimulations. Data points represent means+S.E.M. of n= 8-14 experiments. Area under the carbachol concentration-response curves (AUC) were calculated and compared with

two way ANOVA (p=0.0085 LPS-treated TRPV1+/+ vs. LPS-treated TRPV1-/- mice; p=0.012 TRPV1+/+, LPS vs. TRPV1+/+, LPS+SOM-14; p=0.0062 TRPV1+/+, LPS vs. TRPV1+/+, LPS+C-SOM and p=0.0014 TRPV1-/-, LPS vs. TRPV1-/-, LPS+SOM-14).

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Fig. 6. Role of somatostatin in endotoxin-induced inflammatory histological changes LPS-induced inflammatory changes in lung samples obtained from TRPV1+/+ (A), TRPV1-/- (B), cyclo-somatostatin treated TRPV1+/+ (C) and somatostatin-14-treated TRPV1-/-

(D) mice. Staining was performed with periodic acid-Schiff (PAS) for better identification of mucus producing bronchial goblet cells and shown at 200x magnification; a: alveolar

spaces, b: bronchi, v: vessels.

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Fig. 7. Composite histopathological scores demonstrating the role of somatostatin in endotoxin-induced inflammation Semiquantitative evaluation and scoring of the lung samples on the basis of perivascular oedema formation, perivascular/peribronchial

granulocyte accumulation, goblet cell hyperplasia and alveolar mononuclear cell infiltration. Each column represents the means+S.E.M. of n= 8-14 mice; *p<0.05 TRPV1-

/- vs. TRPV1+/+ group; #p<0.05 cyclo-somatostatin (C-SOM)-treated vs. TRPV1+/+; +p<0.05 somatostatin-14 (SOM-14)-treated vs. respective control group (Kruskal-Wallis

followed by Dunn's test).

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Fig. 8. Role of somatostatin in endotoxin-induced granulocyte accumulation in the lung Myeloperoxidase (MPO) activity, as a quantitative indicator of the number of accumulated

granulocytes, determined from homogenized lung samples 25h after intranasal administration of LPS. The effect of cyclo-somatostatin (C-SOM) treatment in TRPV1+/+

mice and somatostatin-14 (SOM-14) administration in both TRPV1+/+ and TRPV1-/- animals are shown. Results are means+S.E.M. of n= 8-14 mice; *p<0.01 TRPV1-/- vs.

TRPV1+/+ group; #p<0.01 cyclo-somatostatin (C-SOM)-treated vs. TRPV1+/+; +p<0.05; ++p<0.01 somatostatin-14 (SOM-14)-treated vs. respective control group (Kruskal-

Wallis followed by Dunn's test).

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Role of transient receptor potential vanilloid 1 receptors in endotoxin-induced airway inflammation...

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