Alenmyr Et Al-2011-Clinical Physiology and Functional Imaging

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TRPV1 and TRPA 1 stimulation induces MUC5B secretionin the human nasal airway in vivoLisa Alenmyr1, Annkatrin Herrmann2, Edward D. Högestätt1, Lennart Greiff 3 and Peter M. Zygmunt1

1Clinical Chemistry and Pharmacology, Department of Laboratory Medicine, Lund University, 2Cell and Tissue Biology, Department of Experimental Medical Science,

Lund University, and

3

Department of ORL, Head and Neck Surgery, Skå ne University Hospital, Lund, Sweden

CorrespondenceLennart Greiff, Department of ORL, Head and Neck

Surgery, Skå ne University Hospital, SE-221 85

Lund, Sweden

E-mail: [email protected];

[email protected]

Accepted for publicationReceived 4 May 2011;

accepted 17 June 2011

Key wordsMUC5AC; nasal epithelium; sensory nerves;

transient receptor potential; TRPM8

Summary

Aim: Nasal transient receptor potential vanilloid 1 (TRPV1) stimulation withcapsaicin produces serous and mucinous secretion in the human nasal airway. Theprimary aim of this study was to examine topical effects of various TRP ion channelagonists on symptoms and secretion of specific mucins: mucin 5 subtype AC(MUC5AC) and B (MUC5B).Methods: Healthy individuals were subjected to nasal challenges with TRPV1 agonists(capsaicin, olvanil and anandamide), TRP ankyrin 1 (TRPA1) agonists (cinnamal-dehyde and mustard oil) and a TRP melastatin 8 (TRPM8) agonist (menthol).Symptoms were monitored, and nasal lavages were analysed for MUC5AC andMUC5B, i.e. specific mucins associated with airway diseases. In separate groups of healthy subjects, nasal biopsies and brush samples were analysed for TRPV1 andMUC5B, using immunohistochemistry and RT–qPCR. Finally, calcium responses andciliary beat frequency were measured on isolated ciliated epithelial cells.Results: All TRP agonists induced nasal pain or smart. Capsaicin, olvanil and mustardoil also produced rhinorrhea. Lavage fluids obtained after challenge with capsaicinand mustard oil indicated increased levels of MUC5B, whereas MUC5AC wasunaffected. MUC5B and TRPV1 immunoreactivities were primarily localized tosubmucosal glands and peptidergic nerve fibres, respectively. Although trpv1transcripts were detected in nasal brush samples, functional responses to capsaicin

could not be induced in isolated ciliated epithelial cells.Conclusion: Agonists of TRPV1 and TRPA1 induced MUC5B release in the human nasalairways in vivo. These findings may be of relevance with regard to the regulation of mucin production under physiological and pathophysiological conditions.

Introduction

Patients with ongoing seasonal allergic rhinitis may experiencegreater rhinorrhea in response to transient receptor potentialvanilloid 1 (TRPV1) stimulation (e.g. nasal challenge with

capsaicin) compared with asymptomatic patients outside thepollen season (Alenmyr et al., 2009). This likely reflects thehyperresponsiveness that is associated with allergic rhinitis andthat involves mucosal end organs including glands, nerves andblood vessels (Connell, 1969; Druce et al., 1985; Greiff et al.,1995; Svensson et al., 1995). Nasal challenge with capsaicinproduces no or little mucosal exudation of plasma in man(Sanico et al., 1998; Kowalski et al., 1999; Greiff et al., 2005).Thus, rhinorrhea induced by TRPV1 activation is likely to bemediated by the secretory apparatus. This is in agreement withobservations indicating that nasal capsaicin challenge in man

produces increased levels of serous and mucous secretorymarkers in mucosal surface liquids (Geppetti et al., 1988; Philipet al., 1994; Greiff et al., 2005).

Serous and mucous fluid components are two facets of thesecretory apparatus that contributes to physical (e.g. viscoelas-

tic) and bioactive properties of mucosal surface liquids andtherefore to non-specific innate defence actions (Fahy & Dickey,2010). In relation to nasal (and bronchial) pathological airwayconditions, mucin 5 subtype AC (MUC5AC) and B (MUC5B)have received particular attention (Meezaman et al., 1994;Turner & Jones, 2009; Fahy & Dickey, 2010). In normal nasaltissue, mucosal glands show abundant immunostaining forMUC5B, while staining for MUC5AC appears only in goblet cellsof the surface epithelium (Aust et al., 1997; Groneberg et al.,2003). As demonstrated in animals, several signalling moleculesincluding IL-1ß (Fujisawa et al., 2009), IL-13 (Yu et al., 2011),

Clin Physiol Funct Imaging (2011) 31, pp435–444 doi: 10.1111/j.1475-097X.2011.01039.x

2011 The AuthorsClinical Physiology and Functional Imaging 2011 Scandinavian Society of Clinical Physiology and Nuclear Medicine 31, 6, 435–444 435


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IL-17A (Chen et al., 2003; Fujisawa et al., 2009), TNFa (Fischeret al., 1999), EGF (Yuan-Chen Wu et al., 2007; Kim & Nadel,2009), CREB (Kim et al., 2007), fibrinogen ⁄ ICAM-1 (Kim &Nadel, 2009), neutrophil elastase (Kuwahara et al., 2007) andleptin (Woo et al., 2010) may be involved in mucin secretion bygoblet cell and mucosal glands. Yet, little is known about theregulation of airway mucinous secretion in man.

A recent study demonstrated that intratracheal administrationof capsaicin produced mucous secretion and that this responsewas prevented by the TRPV1 antagonist capsazepine (Karmouty-Quintana et al., 2007), implicating TRPV1 as a potentialregulator of airway mucin secretion. Although specific mucinshave been associated with a range of pathological airwayconditions (Fahy & Dickey, 2010) and nasal capsaicin challengehas been demonstrated to produce a global mucosal output of mucins in man (Greiff et al., 2005), little is known about thesecretion of specific mucins in vivo. In this study, using acombined nasal challenge and lavage technique (Greiff et al.,1990), we have examined the effects of TRP ion channel

agonists on symptoms and MUC5AC ⁄ MUC5B secretion inhealthy subjects in vivo. In addition, we have studied the mucosaldistribution of TRPV1 and its association with MUC5B as well asfunctional aspects of TRPV1 in vitro.

Methods

Study design

This study, involving healthy volunteers, examined the effects of nasal challenges with TRPV1, TRP ankyrin 1 (TRPA1) and TRPmelastatin 8 (TRPM8) agonists on symptoms and, in selectedcases, levels of MUC5AC and MUC5B in nasal lavages. The

challenges were carried out in a double-blinded, sham-controlled, randomized and crossover design. Nasal biopsiesobtained from a separate group of subjects were used forimmunohistochemical analysis of TRPV1 and MUC5B as well asthe neuropeptides substance P (SP) and calcitonin gene-relatedpeptide (CGRP) as markers of sensory nerves. Furthermore,nasal brush samples were obtained and analysed for TRPV1using RT–qPCR. Finally, calcium responses and ciliary beatfrequency were measured on freshly isolated ciliated epithelialcells.

Subjects

Fourteen healthy individuals (aged 19–27 years) were subjectedto a panel of sensory nasal challenges. Nasal biopsies wereobtained from four healthy subjects (aged 42–65 years). Brushsamples of the nasal cavity were obtained from a total of tenhealthy subjects (aged 20–30 years): three samples (generatingmultiple cells) were used for functional analysis and seven forRT–qPCR. The study subjects all presented a history withoutindications of upper respiratory tract disease, a normal nasalinspection and a negative skin prick test to seasonal andperennial allergens. Specific exclusion criteria were allergic

rhinitis, structural nasal abnormalities, upper respiratory tractinfections within 14 days before the first visit and preg-nancy ⁄ lactation. No medication was allowed during the study.The study was approved by the regional ethics committee, andinformed consent was obtained from all participants.

ChallengesActive challenges were carried out on 6 days separated by atleast 24 h washout periods. The challenges were delivered aslavages using a pool-device containing 15 ml of fluid, and thefluid was kept in the nasal cavity for 5 min (Greiff et al., 1990).Each active challenge was preceded by two 30-s isotonic salinelavages, carried out to remove mucosal surface liquids thatmight have accumulated for an unknown period of time (theselavages were discarded) and a 5-min sham challenge. On eachday, 1 min elapsed between the sham and active challenges. Therecovered fluids were centrifuged and homogenized, and thealiquots were prepared and frozen ()20C) awaiting analysis.

The active challenges were capsaicin (0Æ5 and 1

Æ0 lmol l

)1

),olvanil (1Æ0 lmol l)1) and anandamide (100 lmol l)1) allacting on TRPV1; cinnamaldehyde (100 lmol l)1) and mustardoil, i.e., allylisothiocyanate, (100 lmol l)1) both acting onTRPA1; and menthol (1Æ0 mmol l)1) acting on TRPM8. Allactive substances were dissolved in isotonic saline with ethanol(1Æ0%), which was also used as sham solution. The dosesselected were based on previous studies (Philip et al., 1994;Greiff et al., 1995, 2005; Sanico et al., 1998; Kowalski et al.,1999; Alenmyr et al., 2009).

Symptoms

The subjects scored symptoms immediately after each shamchallenge and after each active challenge; pain, smart, heat,coolness, itch, rhinorrhea, blockage and lacrimation were eachassigned a score reflecting the severity of the symptom (scorewithin parenthesis): no symptoms (0), mild symptoms (1),moderate symptoms (2) and severe symptoms (3).

Mucin analyses

Lavage fluids obtained after sham challenge and challenge withthe TRPV1 agonist capsaicin (0Æ5 and 1Æ0 lmol l)1) and theTRPA1 agonist mustard oil (100 lmol l)1) were selected for

MUC5AC and MUC5B analyses, as these challenges provokedrhinorrhea. MUC5AC and MUC5B were estimated using anELISA with LUM5-1 and LUM5B-2 antisera, recognizing fullyglycosylated forms of MUC5AC and MUC5B, respectively(Hovenberg et al., 1996; Wickström et al., 1998).

To expose epitopes recognized by LUM5B-2, samples wereinitially reduced by 10 mmol l)1 DTT and alkylated by25 mmol l)1 iodoacetamide prior to analysis (Herrmann et al.,1999). Samples (100 ll), diluted in GuHCl (4Æ0 mol l)1), werecoated onto multiwell assay plates overnight at 4C, followed byblocking (200 ll) with PBS containing 0Æ5% (w ⁄ v) bovine

TRPV1 ⁄ TRPA1-mediated nasal MUC5B secretion, L. Alenmyr et al.

2011 The AuthorsClinical Physiology and Functional Imaging 2011 Scandinavian Society of Clinical Physiology and Nuclear Medicine 31, 6, 435–444

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serum albumin and 0Æ05% (v ⁄ v) Tween 20 for 1 h at roomtemperature. The wells were incubated for 1 h with LUM5-1 orLUM5B-2 antiserum (1:2000 and 1:1000, respectively) inblocking solution. Reactivity was detected with an alkalinephosphatase-conjugated secondary antibody (swine anti-rabbit1:2000 in blocking solution) and detected as absorbance at405 nm after 1 h of incubation with nitrophenyl phosphate

(2 mg ml)1 in 1 mol l)1 diethanolamine–HCl buffer, pH 9Æ8).

Immunohistochemistry

Nasal biopsies were taken from the inferior turbinate, using apair of forceps with a drilled-out punch. Topical anaesthesia andmucosal decongestion was achieved using topical anaesthetics,initially delivered by a spray device and thereafter by a cottonswab. The specimens were fixed in paraformaldehyde (2 Æ0%)and picric acid (0Æ2%) in phosphate-buffered saline (pH 7Æ2 at4C) for 24 h, cryo-protected by rinsing in PBS with sucrose(15%) for 2 days with exchange to fresh solution twice daily,

mounted in OCT-compound, frozen and stored at )

70C.Epithelial cells were obtained from the nasal cavity, using a 6-

mm interdental brush (Apoteket, Stockholm, Sweden). Thesamples were lightly triturated in a physiological buffer solutionafter which 20 ll was smeared on a Superfrost Plus glass slide(Menzel-Gläser, Braunschweig, Germany), air-dried and fixedfor 5 min (fixation as described earlier).

Cryostat sections (20 lm) were processed for the demon-stration of TRPV1, MUC5B, substance P and calcitonin gene-related peptide using indirect immunofluorescence (Table 1).Prior to treatment with the MUC5B antisera (LUM5B-2),sections were reduced and alkylated, respectively, with10 mmol l)1 DTT and 25 mmol l)1 iodoacetamide in0Æ1 mol l)1 Tris ⁄ HCl buffer (pH 8Æ0) for 30 min at roomtemperature. Sections were pretreated with BSA (1%) and TritonX-100 (0Æ2%) for 2 h and incubated with LUM5B-2 for 1 h,followed by incubation with the TRPV1 antiserum overnight atroom temperature. To detect co-localization, cocktails of TRPV1and substance P or calcitonin gene-related peptide antisera wereused. The specimens were then incubated with the secondaryantibodies (Molecular Probes, Eugene, OR, USA) at a dilution of 1:800 for 1 h. When two secondary antibodies were used,incubations were made in sequence. The sensitivity of theantiserum to detect TRPV1 was verified on HEK293 cellstransfected with a plasmid encoding the human TRPV1, using

vector-transfected cells as controls. Control experiments onnative tissue were carried out in the absence of the primary

antibody. In addition, tissue sections were exposed to the TRPV1antiserum preincubated with surplus of blocking peptide.

Digital photographs of the preparations were obtained eitherthrough an Olympus UMPH microscope equipped withNomarski and fluorescence optics with an attached digital sightcamera and a computer equipped with the software Nikon NIS-elements BR 3.0 or with a Nikon Eclipse TE2000-S confocal laser

scanning microscope. Maximum projection images of theconfocal section series were acquired with the software programEZ-C1 Gold version 3.0 (Nikon, Tokyo, Japan). AdobePhotoshop 7.0 (Adobe Systems, San Jose, CA, USA) was usedfor the processing of images.

RT–qPCR

Total RNA was extracted from nasal brushings with Trizolreagent (Invitrogen, Carlsbad, CA, USA) and Nucleospin RNAclean-up (Macherey-Nagel, Düren, Germany), according to themanufacturers instructions. RNA (100 ng) was subjected to

reverse transcription to cDNA using a RT kit (Fermentas K1632Reverse Aid H Minus cDNA kit; Thermo Fisher Scientific,Waltham, MA, USA). Real-time fluorescence-monitored PCRswere performed using TaqMan Universal PCR Master Mix andTaqMan Gene Expression Assay for TRPV1 (Hs00218912_m1;Applied Biosystems, Carlsbad, CA, USA). Thermocycling andreal-time detection of PCR products were performed on aniCyclerIQ sequence detection system (Mx3000P; Stratagene, La

Jolla, CA, USA) with standard cycling parameters. Genes of interest were normalized to the geometric means of tworeference genes, Ubiquitin C (UBC) and glyceraldehyde3-phosphate dehydrogenase (GAPDH), and quantified accord-ing to the DCT method. Reactions were carried out in duplicate.Negative controls consisted of reactions where cDNA wassubstituted with RNA- or nuclease-free water. CT values below37 were considered positive.

Fluorometric calcium imaging

Brush samples from the nasal cavity were obtained from threehealthy individuals. The specimens were suspended in kerati-nocyte serum-free growth medium (Gibco, Invitrogen) con-taining penicillin ⁄ streptomycin (1%) and lightly triturated. Theepithelial cells were dispersed on 8-well slides (Ibidi), coatedwith human placental collagen type IV (Sigma, St. Louis, MO,

USA) and incubated for 18 h at 37C. The cells were incubatedwith Fluo 4-AM (2 lmol l)1) for 15 min at room temperature.

Table 1 Antibodies used in the immunohistochemistry experiments. Rb, Gp and Gt, respectively, denote rabbit, guinea pig and goat. CGRP and SPdenote calcitonin gene-related peptide and substance P, respectively.

Antibody Dilution Code Supplier Host Secondary antibody

TRPV1 1:800 PA1-748 Pierce Biotech., Rockford, IL Rb, polyclonal Alexa Fluor 555 gt anti-rb IgGMUC5B 1:2000 LUM5B-2 Mucosal Biology Group, Lund University, S Rb, antiserum Alexa Fluor 555 gt anti-rb IgGCGRP 1:20 000 B-GP 470-1 Euro-Diagnostica, Malmö, S Gp, antiserum Alexa Fluor 488 gt anti-gp IgGSP 1:2500 B-GP 450-1 Euro-Diagnostica, Malmö, S Gp, antiserum Alexa Fluor 488 gt anti-gp IgG



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The cells were then washed with physiological buffer solutioncontaining (in mmol l)1) 140 NaCl, 5 KCl, 10glucose, 10HEPES,2 CaCl2 and 1 MgCl2 and allowed to equilibrate for a period of 30 minin the dark before the start of the experiments. Changes inintracellular calcium concentration ([Ca2+]i) were monitored atroom temperature using Nikon Eclipse TE2000-S microscope andthe software program EZ-C1 Gold version 3.0 (Nikon).

Ciliary beat frequency assay

The brush samples were lightly triturated in the physiologicalbuffer solution and dispersed in Ibidi chambers as describedearlier. Ciliated cells were studied at room temperature andvisualized with a microscope (Eclipse TE2000-S; Nikon). Thebeat frequency was recorded on a digital high-speed videocamera (5560 HS Endocam, Richard-Wolf, Germany) at a rateof 2000 frames per second. Single cells with synchronouslybeating cilia and a CBF above 5 Hz were identified and analysed.The camera allowed video sequences to be recorded and played

back at reduced frame rates or frame-by-frame. The number of frames required to complete 10 cycles was recorded andconverted to ciliary beat frequency (CBF) according to theequation CBF = 2000 ⁄ (number frames for 10 beats) · 10.

Statistics

Symptoms and DCT values are given as median levels withinterquartile ranges (IQR). Levels of MUC5AC and MUC5B areexpressed as absorption levels (mean ± SEM) at differentdilutions. The areas under the curves (AUCs) were calculatedand displayed separately. Wilcoxon signed rank test was used tocompare symptoms and AUCs for MUC5B and MUC5ACfollowing active and sham challenges. Data on CBF are givenas mean ± SEM. One-tailed paired Students t-test on log-transformed values was used to analyse changes in CBF. P-values


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Immunohistochemistry

MUC5B was localized exclusively to submucosal glands(Fig. 2a). There was no evidence of MUC5B in goblet cells of the epithelium. TRPV1 was detected on nerve fibres aroundblood vessels and mucosal glands (Fig. 2d,e). TRPV1-positivenerve fibres were occasionally found in the epithelium adjacentto MUC5B-expressing structures, probably representing glan-dular ducts (Fig. 2g,h). TRPV1-immunoreactive nerve fibresclose to mucosal glands were co-localized with substance P(Fig. 3a–c) and calcitonin gene-related peptide (Fig. 3d–f).Epithelial nerve fibres expressing TRPV1 did not always displayimmunoreactivity to substance P and calcitonin gene-related

peptide (data not shown).TRPV1 immunoreactivity was recently demonstrated in the

human nasal epithelium (Seki et al., 2006). Using a differentantibody, we could confirm immunostaining of epithelial cellsin both biopsies and nasal brush specimens (Fig. 4). Theimmunofluorescence was condensed at the apical surface of theepithelial cells. No staining was observed in samples exposed tothe secondary antibody only (Fig. 2c). However, preincubationof the TRPV1 antibody with blocking peptide did not removethe immunofluorescence indicating TRPV1-independent bind-ing of the polyclonal antibody (Fig. 4).

RT–qPCR

Analysis of nasal brush samples indicated that trpv1 transcriptswere expressed in the nasal epithelial cells from all examinedindividuals ( n = 7). The CT values for trpv1 and the housekeep-ing gene transcripts were 27Æ0 (range, 26Æ6–28Æ7) and 17Æ9(range, 17Æ8–18Æ9), respectively. The corresponding DCT valuefor the trpv1 transcript was 9Æ3 (range, 8Æ8–10Æ1).

In vitro functional assays

Ciliary epithelial cells from three healthy subjects were used toassess TRPV1 activity using fluorometric calcium imaging and

recording of ciliary beat frequency assays. Capsaicin at aconcentration of 1, 5 or 10 lmol l)1 had no effect on theseparameters in nine tested cells, while ionomycin (1– 10 lmol l)1) and forskolin (50 lmol l)1) produced robustcalcium responses and beat frequency increases, respectively(Fig. 5).

Discussion

This study, focusing on lavage fluid materials obtained fromhealthy subjects, shows that TRPV1 and possibly also TRPA1

(a)

(b)

(c)

Figure 1 ELISA absorbance levels at 405 nm (A405) for MUC5B and MUC5AC in dilution series of lavage samples obtained at challenge with capsaicin(CAP) 0Æ5 lmol l)1 (a), capsaicin 1Æ0 lmol l)1 (b), mustard oil (MO) 100 lmol l)1 (c) and corresponding sham controls. Data are also presented asarea under curve (AUC). MUC5B secretion was increased in response to capsaicin whereas MUC5AC was unaffected. Mustard oil induced a minorMUC5B secretion. *P


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activation produces MUC5B release in the human nasal airwaysin vivo most likely via the stimulation of sensory nerve fibres.The finding may be of relevance with regard to the regulationof airway mucin production and potentially to pathologicalconditions characterized by increased mucus production

including allergic rhinitis, nasal polyposis and chronic rhinos-inusitis.

We examined the effects of a range of agonists targetingdifferent TRP ion channels, including TRPV1, TRPA1 andTRPM8, which are all expressed on primary sensory neurons in

(a) (b)

(c)

(f)(e)(d)

(i)(h)(g)

Figure 2 TRPV1 and MUC5B immunoreac-tivities in nasal biopsies obtained from healthysubjects. Strong MUC5B reactivity was found insubmucosal glands (overlay of Nomarski andfluorescence, 10· magnification, scale bar

equals 100 lm) (a). Weak background stainingin submucosal tissue (b) and epithelium (c)without primary antibodies. Co-staining of MUC5B and TRPV1 revealed TRPV1-immuno-reactive nerve fibres (arrows) adjacent tosubmucosal glands (60· magnification, scalebar equals 50 lm) (d). TRPV1 containingnerves (arrows) were also found within theepithelium of excretory ducts (60· magnifica-tion, scale bar equals 50 lm) (g). Immuno-staining experiments with either TRPV1 (e, h)or MUC5B (f, i) showed the same distributionpattern in submucosal tissue (e, f) and epithe-lium (h, i).

(a) (b) (c)

(d) (e) (f)

Figure 3 TRPV1-immunoreactive nerve fibressurrounding submucosal glands were exploredfor co-localization with the sensory neuropep-tides substance P and calcitonin gene-relatedpeptide (arrows). TRPV1-reactive (a) andSP-reactive (b) nerve fibres co-localized, as

shown in the merged image (c). TRPV1-immunoreactive nerves fibres (d) alsoco-localized with CGRP (e), as shown in themerged image (f). Magnification 60·, scale barequals 50 lm.



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the airways (Nassini et al., 2010). They all produced theexpected profiles of symptom, considering the distribution of the various TRP ion channels on different subpopulations of

sensory neuron. The TRPV1 agonists capsaicin, olvanil andanandamide as well as the TRPA1 agonists mustard oil andcinnamaldehyde, which target a subpopulation of TRPV1-expressing sensory neurons, generally evoked a sensation of heat, pain or smart. The strongest agonists (capsaicin andmustard oil) also induced lacrimation and rhinorrhea. TheTRPM8 agonist menthol produced not only a sensation of coolness but also pain, smart and lacrimation, which are notobviously reconciled with an action on TRPM8. However,menthol can also produce a transient TRPA1 activation and asubpopulation of TRPV1-immunoreactive small-diameter sen-

sory neurons (presumably nociceptive) also expresses TRPM8(Abe et al., 2005; Karashima et al., 2007; Takashima et al., 2010).

Specific mucins currently receive attention in relation to avariety of nasal and bronchial pathological airway conditions.Using a human mucin-secreting goblet cell line, Smirnova et al.(2003) reported that LPS upregulated mRNA expression as wellas production of MUC5AC and MUC5B, suggesting a mecha-nism by which bacterial infections could induce mucinoussecretion. Further, in relation to nasal airway disease, observa-tions in vitro suggested that the expression of MUC5AC andMUC5B, but not that of MUC2 and MUC18, was increased innasal polyposis and allergic rhinitis compared to healthyindividuals (Ji & Guo, 2009). In agreement, increased expres-

sion of MUC5AC and MUC5B was reported in chronicrhinosinusitis and nasal polyposis (Kim et al., 2004; Ali et al.,2005; Viswanathan et al., 2006; Ding & Zheng, 2007).Furthermore, elevated levels of MUC5AC and MUC5B weredemonstrated in sputum or bronchoalveolar lavage fluidsobtained from patients with asthma and COPD (Turner &

Jones, 2009). The present study describes MUC5B secretionafter topical nasal TRPV1 and TRPA1 stimulation in man. Thestrength of these observations lies in the in vivo approach and inthe fact that MUC5B was measured rather than its correspondingmRNA or tissue expression. The present findings indicate that

(a)

(b) (c)

Figure 4 TRPV1 immunoreactivity in epithelial cells. Staining wasfound in parts of the epithelial layers of nasal biopsies from healthysubjects (a). Single ciliated epithelial cells obtained by nasal brushing inhealthy subjects displayed TRPV1 immunoreactivity (b). However, thestaining was not eliminated after preincubation of the TRPV1 antibodywith blocking peptide (c), although a pronounced string of TRPV1-immunoreactivity (b, arrow) disappeared.

(b)

(a)

Figure 5 Lack of functional TRPV1 responses in ciliated epithelial cellsas demonstrated by (a) calcium imaging and (b) recording of ciliarybeat frequency. (a) Cells loaded with the calcium fluorophore fluo-4were exposed to capsaicin (1–10 lmol l)1) followed by exposure to thecalcium–ionophoreionomycin ( n = 9 cells). The calcium signals before(basal) and after application of capsaicin were not different, whereasionomycin always caused a substantial increase in the calcium signal. (b)The adenylate cyclase agonist forskolin, but not capsaicin, increased theciliary beat frequency (CBF) in single ciliated epithelial cells ( n = 4cells). *P


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TRPV1 and possibly also TRPA1 can regulate airway MUC5Bproduction and that these ion channels, as well as other targetsfor pro-inflammatory agents on capsaicin-sensitive nerves, maymediate secretory effects in a wide range of conditionscharacterized by MUC5B production including allergic rhinitis,nasal polyposis, chronic rhinosinusitis, asthma, COPD and cysticfibrosis.

The presence of TRPV1 in the human nasal mucosa was firstreported to be associated with nerve fibres, vascular endothe-lium, submucosal glands and epithelial cells (Seki et al., 2006).However, a subsequent study could only detect TRPV1 on nervefibres (OHanlon et al., 2007). In this study, using a differentestablished antibody (Axelsson et al., 2009), we found evidenceof TRPV1 on peptidergic nerve fibres and epithelial cells. TheTRPV1 immunoreactivity in the epithelial layer of nasal biopsiesindicated that ciliated cells expressed TRPV1. Indeed, we found astrong TRPV1 immunoreactivity on single ciliated epithelial cellsretrieved from nasal brush samples. Although a TRPV1-blockingpeptide could not eliminate the staining of epithelial cells, there

was a marked apical string of TRPV1 immunoreactivity that

disappeared. Therefore, it is possible that TRPV1 is expressed inciliated epithelial cells, which is supported by our finding of trpv1 transcripts in nasal brush samples. As TRPV1 is a non-selective cation channel with high permeability to calcium, itsactivation leads to an increase in intracellular calcium, whichtogether with the second messengers cAMP and cGMP regulatesciliary beat frequency in airway epithelial cells and causes releaseof signalling molecules including cytokines that may activateprimary sensory afferents (Veronesi et al., 1999; Lumpkin &Caterina, 2007; Seki et al., 2007; Braiman & Priel, 2008). In thisstudy, using calcium imaging and recording of ciliary beatfrequency as assays to monitor TRPV1 activity, we did not findany functional TRPV1 responses in single ciliated epithelial cells.This is in line with no effect of capsaicin on cytokine release inhuman primary bronchial epithelial cells (Brandelius et al.,2011). It is also consistent with observations in rodents in vivo,indicating that capsaicin-induced increases in ciliary beatfrequency are neurally mediated (Lindberg & Mercke, 1986).Although a preliminary PCR analysis of cDNA from nasal brushsamples indicated transcription of the full-length hTRPV1(unpublished observations), we cannot exclude the expressionof a splice variant of TRPV1, such as TRPV1b, which negativelyregulates TRPV1 activity (Vos et al., 2006). Clearly, the role of TRPV1 or possible splice variants in human nasal epithelial cell

function remains to be explored.In agreement with reports by Aust et al. (1997) and

Groneberg et al. (2003), we observed MUC5B expressionprimarily localized to mucosal glands. The presence of TRPV1on sensory nerves and its absence on goblet cells andsubmucosal glands is in agreement with the notion thatcapsaicin does not exert direct effects on the latter structures(Chen & Kuo, 1998), but rather allows for reflex-mediatedmucin secretion that is triggered by activation of TRPV1 onsensory nerve fibres. Activation of TRPV1 on sensory nerve

fibres may produce nasal secretion via several mechanisms. Acentral reflex pathway was demonstrated by Philip et al. (1994),who observed contralateral nasal secretion following unilateralcapsaicin challenge (Philip et al., 1994). However, activation of neuronal TRPV1 may also release sensory neurotransmitters,including SP, from the same nerve terminal or from adjacentnerve endings via an axon reflex. Substance P induces the

secretion of mucous glycoproteins from the human nasalmucosa in vitro (Mullol et al., 1992), and its cognate receptorNK1 is expressed on airway submucosal glands and epithelialcells (Shirasaki et al., 1998). In addition, activation of nocicep-tive nerve fibres with hypertonic saline was reported to inducemucin secretion via substance P release, suggesting thatactivation of glandular NK1 receptors may be of importance(Baraniuk et al., 1999). The present findings of TRPV1-positivepeptidergic nerve fibres close to MUC5B-positive submucosalglands provide an anatomical substrate for such a neuroglan-dular interaction in the human nasal mucosa.

Neurogenic inflammation, typically demonstrated as capsa-

icin-induced plasma exudation, is well recognized in rodents(Erjefält & Persson, 1985). Although the role of sensory neuronsin human airway inflammation is less clear (Greiff et al., 1995),this and previous studies unequivocally show that these neuronsmediate some of the signs and symptoms, including itch,sneezes and reflex-mediated secretion, typically encounteredduring allergic rhinitis (Raphael et al., 1991). Interestingly, thesensory responsiveness to capsaicin tends to be amplified inseasonal allergic rhinitis (Greiff et al., 1995; Kowalski et al.,1999; Alenmyr et al., 2009). This study corroborates thesefindings and further shows that activation of TRPV1 andpossibly also of TRPA1 produces the secretion of MUC5B, amucin specifically associated with inflammatory airway disease(see above).

In conclusion, we demonstrate that activation of nasal TRPV1and possibly also TRPA1 produces rhinorrhea and secretion of MUC5B from the human nasal airway in vivo. Although ourimmunohistochemial findings are compatible with a neuro-glandular interaction, additional functional studies are requiredto sort out the relative contribution of reflex-dependent andreflex-independent pathways. Further studies are also warrantedto explore the role of capsaicin-sensitive sensory neurons as wellas TRPV1 and TRPA1 as treatment targets in nasal airwayconditions associated with excessive mucosal secretion.

Acknowledgments

The study was supported by grants from the Swedish ResearchCouncil (2007-3095), the Swedish Cancer and Allergy Foun-dation, the Swedish Asthma and Allergy Foundation and LundUniversity (ALF). We thank Dr. Eva Millqvist (GothenburgUniversity) for providing nasal biopsies and Professor MartinKanje (Lund University) for help with the Nomarski analysis.We also thank Charlotte Cervin-Hoberg, Christina Falk Olssonand Lena Glantz-Larsson for laboratory assistance.



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