Clin Physiol Funct Imaging (2011) 31, pp435444

doi: 10.1111/j.1475-097X.2011.01039.x

TRPV1 and TRPA1 stimulation induces MUC5B secretion

in the human nasal airway in vivo
gestatt1, Lennart Greiff3 and Peter M. Zygmunt1
Lisa Alenmyr1, Annkatrin Herrmann2, Edward D. Ho
1

Clinical Chemistry and Pharmacology, Department of Laboratory Medicine, Lund University, 2Cell and Tissue Biology, Department of Experimental Medical Science,
Lund University, and 3Department of ORL, Head and Neck Surgery, Skane University Hospital, Lund, Sweden

Summary
Correspondence
Lennart Greiff, Department of ORL, Head and Neck
Surgery, Skane University Hospital, SE-221 85
Lund, Sweden
E-mail: lennart.greiff@skane.se;
lennart.greiff@live.se

Accepted for publication

Received 4 May 2011;
accepted 17 June 2011

Key words
MUC5AC; nasal epithelium; sensory nerves;
transient receptor potential; TRPM8

Aim: Nasal transient receptor potential vanilloid 1 (TRPV1) stimulation with

capsaicin produces serous and mucinous secretion in the human nasal airway. The
primary aim of this study was to examine topical effects of various TRP ion channel
agonists 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 (cinnamaldehyde and mustard oil) and a TRP melastatin 8 (TRPM8) agonist (menthol).
Symptoms were monitored, and nasal lavages were analysed for MUC5AC and
MUC5B, i.e. specific mucins associated with airway diseases. In separate groups of
healthy subjects, nasal biopsies and brush samples were analysed for TRPV1 and
MUC5B, using immunohistochemistry and RTqPCR. Finally, calcium responses and
ciliary beat frequency were measured on isolated ciliated epithelial cells.
Results: All TRP agonists induced nasal pain or smart. Capsaicin, olvanil and mustard
oil also produced rhinorrhea. Lavage fluids obtained after challenge with capsaicin
and mustard oil indicated increased levels of MUC5B, whereas MUC5AC was
unaffected. MUC5B and TRPV1 immunoreactivities were primarily localized to
submucosal glands and peptidergic nerve fibres, respectively. Although trpv1
transcripts 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 nasal
airways 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 experience
greater rhinorrhea in response to transient receptor potential
vanilloid 1 (TRPV1) stimulation (e.g. nasal challenge with
capsaicin) compared with asymptomatic patients outside the
pollen season (Alenmyr et al., 2009). This likely reflects the
hyperresponsiveness that is associated with allergic rhinitis and
that involves mucosal end organs including glands, nerves and
blood vessels (Connell, 1969; Druce et al., 1985; Greiff et al.,
1995; Svensson et al., 1995). Nasal challenge with capsaicin
produces 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 be
mediated by the secretory apparatus. This is in agreement with
observations indicating that nasal capsaicin challenge in man

produces increased levels of serous and mucous secretory

markers in mucosal surface liquids (Geppetti et al., 1988; Philip
et al., 1994; Greiff et al., 2005).
Serous and mucous fluid components are two facets of the
secretory apparatus that contributes to physical (e.g. viscoelastic) and bioactive properties of mucosal surface liquids and
therefore to non-specific innate defence actions (Fahy & Dickey,
2010). In relation to nasal (and bronchial) pathological airway
conditions, mucin 5 subtype AC (MUC5AC) and B (MUC5B)
have received particular attention (Meezaman et al., 1994;
Turner & Jones, 2009; Fahy & Dickey, 2010). In normal nasal
tissue, mucosal glands show abundant immunostaining for
MUC5B, while staining for MUC5AC appears only in goblet cells
of the surface epithelium (Aust et al., 1997; Groneberg et al.,
2003). As demonstrated in animals, several signalling molecules
including IL-1 (Fujisawa et al., 2009), IL-13 (Yu et al., 2011),

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435

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

IL-17A (Chen et al., 2003; Fujisawa et al., 2009), TNFa (Fischer

et 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) and
leptin (Woo et al., 2010) may be involved in mucin secretion by
goblet cell and mucosal glands. Yet, little is known about the
regulation of airway mucinous secretion in man.
A recent study demonstrated that intratracheal administration
of capsaicin produced mucous secretion and that this response
was prevented by the TRPV1 antagonist capsazepine (KarmoutyQuintana et al., 2007), implicating TRPV1 as a potential
regulator of airway mucin secretion. Although specific mucins
have been associated with a range of pathological airway
conditions (Fahy & Dickey, 2010) and nasal capsaicin challenge
has been demonstrated to produce a global mucosal output of
mucins in man (Greiff et al., 2005), little is known about the
secretion of specific mucins in vivo. In this study, using a
combined 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 in
healthy subjects in vivo. In addition, we have studied the mucosal
distribution of TRPV1 and its association with MUC5B as well as
functional 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 TRP
melastatin 8 (TRPM8) agonists on symptoms and, in selected
cases, levels of MUC5AC and MUC5B in nasal lavages. The
challenges were carried out in a double-blinded, shamcontrolled, randomized and crossover design. Nasal biopsies
obtained from a separate group of subjects were used for
immunohistochemical analysis of TRPV1 and MUC5B as well as
the neuropeptides substance P (SP) and calcitonin gene-related
peptide (CGRP) as markers of sensory nerves. Furthermore,
nasal brush samples were obtained and analysed for TRPV1
using RTqPCR. Finally, calcium responses and ciliary beat
frequency were measured on freshly isolated ciliated epithelial
cells.
Subjects
Fourteen healthy individuals (aged 1927 years) were subjected
to a panel of sensory nasal challenges. Nasal biopsies were
obtained from four healthy subjects (aged 4265 years). Brush
samples of the nasal cavity were obtained from a total of ten
healthy subjects (aged 2030 years): three samples (generating
multiple cells) were used for functional analysis and seven for
RTqPCR. The study subjects all presented a history without
indications of upper respiratory tract disease, a normal nasal
inspection and a negative skin prick test to seasonal and
perennial allergens. Specific exclusion criteria were allergic

rhinitis, structural nasal abnormalities, upper respiratory tract

infections within 14 days before the first visit and pregnancy lactation. No medication was allowed during the study.
The study was approved by the regional ethics committee, and
informed consent was obtained from all participants.
Challenges
Active challenges were carried out on 6 days separated by at
least 24 h washout periods. The challenges were delivered as
lavages using a pool-device containing 15 ml of fluid, and the
fluid was kept in the nasal cavity for 5 min (Greiff et al., 1990).
Each active challenge was preceded by two 30-s isotonic saline
lavages, carried out to remove mucosal surface liquids that
might have accumulated for an unknown period of time (these
lavages were discarded) and a 5-min sham challenge. On each
day, 1 min elapsed between the sham and active challenges. The
recovered fluids were centrifuged and homogenized, and the
aliquots were prepared and frozen ()20C) awaiting analysis.
The active challenges were capsaicin (05 and 10 lmol l)1),
olvanil (10 lmol l)1) and anandamide (100 lmol l)1) all
acting on TRPV1; cinnamaldehyde (100 lmol l)1) and mustard
oil, i.e., allylisothiocyanate, (100 lmol l)1) both acting on
TRPA1; and menthol (10 mmol l)1) acting on TRPM8. All
active substances were dissolved in isotonic saline with ethanol
(10%), which was also used as sham solution. The doses
selected 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 sham
challenge and after each active challenge; pain, smart, heat,
coolness, itch, rhinorrhea, blockage and lacrimation were each
assigned a score reflecting the severity of the symptom (score
within parenthesis): no symptoms (0), mild symptoms (1),
moderate symptoms (2) and severe symptoms (3).
Mucin analyses
Lavage fluids obtained after sham challenge and challenge with
the TRPV1 agonist capsaicin (05 and 10 lmol l)1) and the
TRPA1 agonist mustard oil (100 lmol l)1) were selected for
MUC5AC and MUC5B analyses, as these challenges provoked
rhinorrhea. MUC5AC and MUC5B were estimated using an
ELISA with LUM5-1 and LUM5B-2 antisera, recognizing fully
glycosylated forms of MUC5AC and MUC5B, respectively
(Hovenberg et al., 1996; Wickstrom et al., 1998).
To expose epitopes recognized by LUM5B-2, samples were
initially reduced by 10 mmol l)1 DTT and alkylated by
25 mmol l)1 iodoacetamide prior to analysis (Herrmann et al.,
1999). Samples (100 ll), diluted in GuHCl (40 mol l)1), were
coated onto multiwell assay plates overnight at 4C, followed by
blocking (200 ll) with PBS containing 05% (w v) bovine

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TRPV1 TRPA1-mediated nasal MUC5B secretion, L. Alenmyr et al. 437

serum albumin and 005% (v v) Tween 20 for 1 h at room

temperature. The wells were incubated for 1 h with LUM5-1 or
LUM5B-2 antiserum (1:2000 and 1:1000, respectively) in
blocking solution. Reactivity was detected with an alkaline
phosphatase-conjugated secondary antibody (swine anti-rabbit
1:2000 in blocking solution) and detected as absorbance at
405 nm after 1 h of incubation with nitrophenyl phosphate
(2 mg ml)1 in 1 mol l)1 diethanolamineHCl buffer, pH 98).
Immunohistochemistry
Nasal biopsies were taken from the inferior turbinate, using a
pair of forceps with a drilled-out punch. Topical anaesthesia and
mucosal decongestion was achieved using topical anaesthetics,
initially delivered by a spray device and thereafter by a cotton
swab. The specimens were fixed in paraformaldehyde (20%)
and picric acid (02%) in phosphate-buffered saline (pH 72 at
4C) 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 6mm interdental brush (Apoteket, Stockholm, Sweden). The
samples were lightly triturated in a physiological buffer solution
after which 20 ll was smeared on a Superfrost Plus glass slide
(Menzel-Glaser, Braunschweig, Germany), air-dried and fixed
for 5 min (fixation as described earlier).
Cryostat sections (20 lm) were processed for the demonstration of TRPV1, MUC5B, substance P and calcitonin generelated peptide using indirect immunofluorescence (Table 1).
Prior to treatment with the MUC5B antisera (LUM5B-2),
sections were reduced and alkylated, respectively, with
10 mmol l)1 DTT and 25 mmol l)1 iodoacetamide in
01 mol l)1 Tris HCl buffer (pH 80) for 30 min at room
temperature. Sections were pretreated with BSA (1%) and Triton
X-100 (02%) for 2 h and incubated with LUM5B-2 for 1 h,
followed by incubation with the TRPV1 antiserum overnight at
room temperature. To detect co-localization, cocktails of TRPV1
and substance P or calcitonin gene-related peptide antisera were
used. The specimens were then incubated with the secondary
antibodies (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 the
antiserum to detect TRPV1 was verified on HEK293 cells
transfected with a plasmid encoding the human TRPV1, using
vector-transfected cells as controls. Control experiments on
native tissue were carried out in the absence of the primary

antibody. In addition, tissue sections were exposed to the TRPV1

antiserum preincubated with surplus of blocking peptide.
Digital photographs of the preparations were obtained either
through an Olympus UMPH microscope equipped with
Nomarski and fluorescence optics with an attached digital sight
camera and a computer equipped with the software Nikon NISelements BR 3.0 or with a Nikon Eclipse TE2000-S confocal laser
scanning microscope. Maximum projection images of the
confocal section series were acquired with the software program
EZ-C1 Gold version 3.0 (Nikon, Tokyo, Japan). Adobe
Photoshop 7.0 (Adobe Systems, San Jose, CA, USA) was used
for the processing of images.
RTqPCR
Total RNA was extracted from nasal brushings with Trizol
reagent (Invitrogen, Carlsbad, CA, USA) and Nucleospin RNA
clean-up (Macherey-Nagel, Duren, Germany), according to the
manufacturers instructions. RNA (100 ng) was subjected to
reverse transcription to cDNA using a RT kit (Fermentas K1632
Reverse Aid H Minus cDNA kit; Thermo Fisher Scientific,
Waltham, MA, USA). Real-time fluorescence-monitored PCRs
were performed using TaqMan Universal PCR Master Mix and
TaqMan Gene Expression Assay for TRPV1 (Hs00218912_m1;
Applied Biosystems, Carlsbad, CA, USA). Thermocycling and
real-time detection of PCR products were performed on an
iCyclerIQ sequence detection system (Mx3000P; Stratagene, La
Jolla, CA, USA) with standard cycling parameters. Genes of
interest were normalized to the geometric means of two
reference genes, Ubiquitin C (UBC) and glyceraldehyde
3-phosphate dehydrogenase (GAPDH), and quantified according to the DCT method. Reactions were carried out in duplicate.
Negative controls consisted of reactions where cDNA was
substituted with RNA- or nuclease-free water. CT values below
37 were considered positive.
Fluorometric calcium imaging
Brush samples from the nasal cavity were obtained from three
healthy individuals. The specimens were suspended in keratinocyte serum-free growth medium (Gibco, Invitrogen) containing penicillin streptomycin (1%) and lightly triturated. The
epithelial cells were dispersed on 8-well slides (Ibidi), coated
with human placental collagen type IV (Sigma, St. Louis, MO,
USA) and incubated for 18 h at 37C. The cells were incubated
with 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 SP
denote calcitonin gene-related peptide and substance P, respectively.
Antibody

Dilution

Code

Supplier

Host

Secondary antibody

TRPV1
MUC5B
CGRP
SP

1:800
1:2000
1:20 000
1:2500

PA1-748
LUM5B-2
B-GP 470-1
B-GP 450-1

Pierce Biotech., Rockford, IL

Mucosal Biology Group, Lund University, S
Euro-Diagnostica, Malmo, S
Euro-Diagnostica, Malmo, S

Rb, polyclonal
Rb, antiserum
Gp, antiserum
Gp, antiserum

Alexa Fluor 555 gt anti-rb IgG

Alexa Fluor 555 gt anti-rb IgG
Alexa Fluor 488 gt anti-gp IgG
Alexa Fluor 488 gt anti-gp IgG

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

The cells were then washed with physiological buffer solution

containing (in mmol l)1) 140 NaCl, 5 KCl, 10 glucose, 10 HEPES,
2 CaCl2 and 1 MgCl2 and allowed to equilibrate for a period of
30 min in the dark before the start of the experiments. Changes in
intracellular calcium concentration ([Ca2+]i) were monitored at
room temperature using Nikon Eclipse TE2000-S microscope and
the software program EZ-C1 Gold version 3.0 (Nikon).
Ciliary beat frequency assay
The brush samples were lightly triturated in the physiological
buffer solution and dispersed in Ibidi chambers as described
earlier. Ciliated cells were studied at room temperature and
visualized with a microscope (Eclipse TE2000-S; Nikon). The
beat frequency was recorded on a digital high-speed video
camera (5560 HS Endocam, Richard-Wolf, Germany) at a rate
of 2000 frames per second. Single cells with synchronously
beating 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 and
converted to ciliary beat frequency (CBF) according to the
equation CBF = 2000 (number frames for 10 beats) 10.
Statistics
Symptoms and DCT values are given as median levels with
interquartile ranges (IQR). Levels of MUC5AC and MUC5B are
expressed as absorption levels (mean SEM) at different
dilutions. The areas under the curves (AUCs) were calculated
and displayed separately. Wilcoxon signed rank test was used to
compare symptoms and AUCs for MUC5B and MUC5AC
following active and sham challenges. Data on CBF are given
as mean SEM. One-tailed paired Students t-test on logtransformed values was used to analyse changes in CBF. Pvalues<005 were considered statistically significant.

Results
Symptoms
Symptoms produced by the challenge with TRPV1 agonists are
presented in Table 2. The symptoms produced by capsaicin

were dominated by pain (both doses: P<0005) and smart

(05 lmol l)1, P<0005; 10 lmol l)1, P<0001). This TRPV1
agonist also produced a sensation of heat (05 lmol l)1,
P<005; 10 lmol l)1, P<0005), rhinorrhea (10 lmol l)1,
P<005) and lacrimation (both doses, P<0005). Olvanil
produced pain (P<005), smart (P<0005) and rhinorrhea
(P<005). Anandamide produced smart (P<0005).
The TRPA1 agonists produced pain and smart (Table 3).
Accordingly, cinnamaldehyde produced smart (P<0005), while
mustard oil caused pain (P<005), smart (P<0005), heat
sensation (P<005), lacrimation (P<001) and rhinorrhea
(P<005). The TRPM8 agonist menthol evoked pain (P<005),
smart (P<0005), cool sensation (P<0005) and lacrimation
(P<005).
Mucin secretion
Using the ELISA technique, serial dilutions of nasal lavage fluids
were analysed for MUC5B and MUC5AC contents (Fig. 1). Both
doses of capsaicin produced an increase in the level of MUC5B
(P<0001, respectively, c.f. sham). In contrast, the level of
MUC5AC was unaffected by capsaicin. The TRPA1 agonist
mustard oil also induced a significant secretion of MUC5B
(P<005, c.f. sham), but this response was less pronounced than
after TRPV1 stimulation. As with capsaicin, mustard oil did not
affect the nasal lavage fluid level of MUC5AC.
Table 3 Effects of challenges with TRPA1 and TRPM8 agonists on
nasal symptoms. The symptom profile was dominated by smart.
Symptom levels of the sham challenge were scored 0 or close to 0 at all
occasions (data not shown). For statistical differences, see the Results
section.
TRPA1
Symptoms
(Score 03)

Cinnamaldehyde
(100 lmol l)1)

Pain
Smart
Heat
Coolness
Itch
Rhinorrhea
Lacrimation

0
1
0
0
0
0
0

TRPV1
Symptoms
(Score 03)
Pain
Smart
Heat
Coolness
Itch
Rhinorrhea
Lacrimation

Capsaicin
(05 lmol l)1)
1
2
1
0
0
0
1

(052)
(12)
(02)
(00)
(00)
(01)
(12)

Capsaicin
(10 lmol l)1)
2
25
2
0
0
05
2

(052)
(23)
(12)
(00)
(00)
(015)
(12)

Olvanil
(10 lmol l)1)
0
1
0
0
0
0
0

(01)
(052)
(01)
(00)
(00)
(01)
(00)

Anandamide
(100 lmol l)1)
0
1
0
0
0
0
0

(00)
(01)
(01)
(00)
(00)
(00)
(00)

(005)
(01)
(01)
(005)
(00)
(01)
(00)

TRPM8

Mustard oil
(100 lmol l)1)
0
15
1
0
0
0
1

(02)
(125)
(01)
(00)
(00)
(01)
(015)

Menthol
(10 mmol l)1)
0
15
0
15
0
0
0

(01)
(052)
(005)
(052)
(00)
(00)
(01)

Table 2 Effects of challenges with TRPV1

agonists on nasal symptoms. The symptom
profile was dominated by smart. In addition,
capsaicin produced pain, a sensation of heat,
secretion and lacrimation. Symptom levels of
the sham challenge were scored 0 or close to 0
at all occasions (data not shown). For statistical
differences, see the Results section.

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TRPV1 TRPA1-mediated nasal MUC5B secretion, L. Alenmyr et al. 439

(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) 05 lmol l)1 (a), capsaicin 10 lmol l)1 (b), mustard oil (MO) 100 lmol l)1 (c) and corresponding sham controls. Data are also presented as
area under curve (AUC). MUC5B secretion was increased in response to capsaicin whereas MUC5AC was unaffected. Mustard oil induced a minor
MUC5B secretion. *P<005 and ***P<0001.

Immunohistochemistry

RTqPCR

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 around
blood vessels and mucosal glands (Fig. 2d,e). TRPV1-positive
nerve fibres were occasionally found in the epithelium adjacent
to MUC5B-expressing structures, probably representing glandular ducts (Fig. 2g,h). TRPV1-immunoreactive nerve fibres
close to mucosal glands were co-localized with substance P
(Fig. 3ac) and calcitonin gene-related peptide (Fig. 3df).
Epithelial nerve fibres expressing TRPV1 did not always display
immunoreactivity 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 different
antibody, we could confirm immunostaining of epithelial cells
in both biopsies and nasal brush specimens (Fig. 4). The
immunofluorescence was condensed at the apical surface of the
epithelial cells. No staining was observed in samples exposed to
the secondary antibody only (Fig. 2c). However, preincubation
of the TRPV1 antibody with blocking peptide did not remove
the immunofluorescence indicating TRPV1-independent binding of the polyclonal antibody (Fig. 4).

Analysis of nasal brush samples indicated that trpv1 transcripts

were expressed in the nasal epithelial cells from all examined
individuals (n = 7). The CT values for trpv1 and the housekeeping gene transcripts were 270 (range, 266287) and 179
(range, 178189), respectively. The corresponding DCT value
for the trpv1 transcript was 93 (range, 88101).
In vitro functional assays
Ciliary epithelial cells from three healthy subjects were used to
assess TRPV1 activity using fluorometric calcium imaging and
recording of ciliary beat frequency assays. Capsaicin at a
concentration of 1, 5 or 10 lmol l)1 had no effect on these
parameters in nine tested cells, while ionomycin (1
10 lmol l)1) and forskolin (50 lmol l)1) produced robust
calcium responses and beat frequency increases, respectively
(Fig. 5).

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

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

(a)

(b)

(c)

(d)

(e)

(g)

(a)

(d)

(f)

(h)

(b)

(e)

(i)

Figure 2 TRPV1 and MUC5B immunoreactivities in nasal biopsies obtained from healthy
subjects. Strong MUC5B reactivity was found in
submucosal glands (overlay of Nomarski and
fluorescence, 10 magnification, scale bar
equals 100 lm) (a). Weak background staining
in submucosal tissue (b) and epithelium (c)
without primary antibodies. Co-staining of
MUC5B and TRPV1 revealed TRPV1-immunoreactive nerve fibres (arrows) adjacent to
submucosal glands (60 magnification, scale
bar equals 50 lm) (d). TRPV1 containing
nerves (arrows) were also found within the
epithelium of excretory ducts (60 magnification, scale bar equals 50 lm) (g). Immunostaining experiments with either TRPV1 (e, h)
or MUC5B (f, i) showed the same distribution
pattern in submucosal tissue (e, f) and epithelium (h, i).

(c)

(f)

activation produces MUC5B release in the human nasal airways

in vivo most likely via the stimulation of sensory nerve fibres.
The finding may be of relevance with regard to the regulation
of airway mucin production and potentially to pathological
conditions characterized by increased mucus production

Figure 3 TRPV1-immunoreactive nerve fibres

surrounding submucosal glands were explored
for co-localization with the sensory neuropeptides substance P and calcitonin gene-related
peptide (arrows). TRPV1-reactive (a) and
SP-reactive (b) nerve fibres co-localized, as
shown in the merged image (c). TRPV1immunoreactive nerves fibres (d) also
co-localized with CGRP (e), as shown in the
merged image (f). Magnification 60, scale bar
equals 50 lm.

including allergic rhinitis, nasal polyposis and chronic rhinosinusitis.

We examined the effects of a range of agonists targeting
different TRP ion channels, including TRPV1, TRPA1 and
TRPM8, which are all expressed on primary sensory neurons in

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Clinical Physiology and Functional Imaging  2011 Scandinavian Society of Clinical Physiology and Nuclear Medicine 31, 6, 435444

TRPV1 TRPA1-mediated nasal MUC5B secretion, L. Alenmyr et al. 441

(a)

(a)

(b)

(b)

(c)
Figure 5 Lack of functional TRPV1 responses in ciliated epithelial cells
as demonstrated by (a) calcium imaging and (b) recording of ciliary
beat frequency. (a) Cells loaded with the calcium fluorophore fluo-4
were exposed to capsaicin (110 lmol l)1) followed by exposure to the
calciumionophoreionomycin (n = 9 cells). The calcium signals before
(basal) and after application of capsaicin were not different, whereas
ionomycin always caused a substantial increase in the calcium signal. (b)
The adenylate cyclase agonist forskolin, but not capsaicin, increased the
ciliary beat frequency (CBF) in single ciliated epithelial cells (n = 4
cells). *P<005.

Figure 4 TRPV1 immunoreactivity in epithelial cells. Staining was

found in parts of the epithelial layers of nasal biopsies from healthy
subjects (a). Single ciliated epithelial cells obtained by nasal brushing in
healthy subjects displayed TRPV1 immunoreactivity (b). However, the
staining was not eliminated after preincubation of the TRPV1 antibody
with blocking peptide (c), although a pronounced string of TRPV1immunoreactivity (b, arrow) disappeared.

the airways (Nassini et al., 2010). They all produced the

expected profiles of symptom, considering the distribution of
the various TRP ion channels on different subpopulations of
sensory neuron. The TRPV1 agonists capsaicin, olvanil and
anandamide as well as the TRPA1 agonists mustard oil and
cinnamaldehyde, which target a subpopulation of TRPV1expressing sensory neurons, generally evoked a sensation of
heat, pain or smart. The strongest agonists (capsaicin and
mustard oil) also induced lacrimation and rhinorrhea. The
TRPM8 agonist menthol produced not only a sensation of
coolness but also pain, smart and lacrimation, which are not
obviously reconciled with an action on TRPM8. However,
menthol can also produce a transient TRPA1 activation and a
subpopulation 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 a
variety 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 well
as production of MUC5AC and MUC5B, suggesting a mechanism by which bacterial infections could induce mucinous
secretion. Further, in relation to nasal airway disease, observations in vitro suggested that the expression of MUC5AC and
MUC5B, but not that of MUC2 and MUC18, was increased in
nasal polyposis and allergic rhinitis compared to healthy
individuals (Ji & Guo, 2009). In agreement, increased expression of MUC5AC and MUC5B was reported in chronic
rhinosinusitis 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 were
demonstrated in sputum or bronchoalveolar lavage fluids
obtained from patients with asthma and COPD (Turner &
Jones, 2009). The present study describes MUC5B secretion
after topical nasal TRPV1 and TRPA1 stimulation in man. The
strength of these observations lies in the in vivo approach and in
the fact that MUC5B was measured rather than its corresponding
mRNA or tissue expression. The present findings indicate that

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Clinical Physiology and Functional Imaging  2011 Scandinavian Society of Clinical Physiology and Nuclear Medicine 31, 6, 435444

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

TRPV1 and possibly also TRPA1 can regulate airway MUC5B

production and that these ion channels, as well as other targets
for pro-inflammatory agents on capsaicin-sensitive nerves, may
mediate secretory effects in a wide range of conditions
characterized by MUC5B production including allergic rhinitis,
nasal polyposis, chronic rhinosinusitis, asthma, COPD and cystic
fibrosis.
The presence of TRPV1 in the human nasal mucosa was first
reported to be associated with nerve fibres, vascular endothelium, submucosal glands and epithelial cells (Seki et al., 2006).
However, a subsequent study could only detect TRPV1 on nerve
fibres (OHanlon et al., 2007). In this study, using a different
established antibody (Axelsson et al., 2009), we found evidence
of TRPV1 on peptidergic nerve fibres and epithelial cells. The
TRPV1 immunoreactivity in the epithelial layer of nasal biopsies
indicated that ciliated cells expressed TRPV1. Indeed, we found a
strong TRPV1 immunoreactivity on single ciliated epithelial cells
retrieved from nasal brush samples. Although a TRPV1-blocking
peptide 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 in
ciliated epithelial cells, which is supported by our finding of
trpv1 transcripts in nasal brush samples. As TRPV1 is a nonselective cation channel with high permeability to calcium, its
activation leads to an increase in intracellular calcium, which
together with the second messengers cAMP and cGMP regulates
ciliary beat frequency in airway epithelial cells and causes release
of signalling molecules including cytokines that may activate
primary sensory afferents (Veronesi et al., 1999; Lumpkin &
Caterina, 2007; Seki et al., 2007; Braiman & Priel, 2008). In this
study, using calcium imaging and recording of ciliary beat
frequency as assays to monitor TRPV1 activity, we did not find
any functional TRPV1 responses in single ciliated epithelial cells.
This is in line with no effect of capsaicin on cytokine release in
human 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 beat
frequency are neurally mediated (Lindberg & Mercke, 1986).
Although a preliminary PCR analysis of cDNA from nasal brush
samples indicated transcription of the full-length hTRPV1
(unpublished observations), we cannot exclude the expression
of a splice variant of TRPV1, such as TRPV1b, which negatively
regulates 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 expression
primarily localized to mucosal glands. The presence of TRPV1
on sensory nerves and its absence on goblet cells and
submucosal glands is in agreement with the notion that
capsaicin does not exert direct effects on the latter structures
(Chen & Kuo, 1998), but rather allows for reflex-mediated
mucin secretion that is triggered by activation of TRPV1 on
sensory nerve fibres. Activation of TRPV1 on sensory nerve

fibres may produce nasal secretion via several mechanisms. A

central reflex pathway was demonstrated by Philip et al. (1994),
who observed contralateral nasal secretion following unilateral
capsaicin challenge (Philip et al., 1994). However, activation of
neuronal TRPV1 may also release sensory neurotransmitters,
including SP, from the same nerve terminal or from adjacent
nerve endings via an axon reflex. Substance P induces the
secretion of mucous glycoproteins from the human nasal
mucosa in vitro (Mullol et al., 1992), and its cognate receptor
NK1 is expressed on airway submucosal glands and epithelial
cells (Shirasaki et al., 1998). In addition, activation of nociceptive nerve fibres with hypertonic saline was reported to induce
mucin secretion via substance P release, suggesting that
activation of glandular NK1 receptors may be of importance
(Baraniuk et al., 1999). The present findings of TRPV1-positive
peptidergic nerve fibres close to MUC5B-positive submucosal
glands provide an anatomical substrate for such a neuroglandular interaction in the human nasal mucosa.
Neurogenic inflammation, typically demonstrated as capsaicin-induced plasma exudation, is well recognized in rodents
(Erjefalt & Persson, 1985). Although the role of sensory neurons
in human airway inflammation is less clear (Greiff et al., 1995),
this and previous studies unequivocally show that these neurons
mediate some of the signs and symptoms, including itch,
sneezes and reflex-mediated secretion, typically encountered
during allergic rhinitis (Raphael et al., 1991). Interestingly, the
sensory responsiveness to capsaicin tends to be amplified in
seasonal allergic rhinitis (Greiff et al., 1995; Kowalski et al.,
1999; Alenmyr et al., 2009). This study corroborates these
findings and further shows that activation of TRPV1 and
possibly also of TRPA1 produces the secretion of MUC5B, a
mucin specifically associated with inflammatory airway disease
(see above).
In conclusion, we demonstrate that activation of nasal TRPV1
and possibly also TRPA1 produces rhinorrhea and secretion of
MUC5B from the human nasal airway in vivo. Although our
immunohistochemial findings are compatible with a neuroglandular interaction, additional functional studies are required
to sort out the relative contribution of reflex-dependent and
reflex-independent pathways. Further studies are also warranted
to explore the role of capsaicin-sensitive sensory neurons as well
as TRPV1 and TRPA1 as treatment targets in nasal airway
conditions associated with excessive mucosal secretion.

Acknowledgments
The study was supported by grants from the Swedish Research
Council (2007-3095), the Swedish Cancer and Allergy Foundation, the Swedish Asthma and Allergy Foundation and Lund
University (ALF). We thank Dr. Eva Millqvist (Gothenburg
University) for providing nasal biopsies and Professor Martin
Kanje (Lund University) for help with the Nomarski analysis.
We also thank Charlotte Cervin-Hoberg, Christina Falk Olsson
and Lena Glantz-Larsson for laboratory assistance.

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TRPV1 TRPA1-mediated nasal MUC5B secretion, L. Alenmyr et al. 443

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Clinical Physiology and Functional Imaging  2011 Scandinavian Society of Clinical Physiology and Nuclear Medicine 31, 6, 435444