Intranasal steroids decrease eosinophils but not mucin expression in
P-R. Burgel*, L.O. Cardell#, I.F. Ueki}, J.A. Nadel}
Intranasal steroids decrease eosinophils but not mucin expression in nasal polyps.
P-R. Burgel, L.O. Cardell, I.F. Ueki, J.A. Nadel. #ERS Journals Ltd 2004.
ABSTRACT: Increased mucin expression is a feature of nasal polyposis. Cortico-
steroids reduce polyp size and symptoms, but their effect on mucin production remains
unknown. In this study, the effects of intranasal corticosteroids on MUC5AC mucin
expression, nasal resistance, eosinophil and neutrophil infiltration, epidermal growth
factor receptor (EGFR), interleukin (IL)-8, and tumour necrosis factor (TNF)-a
expression was assessed in nasal polyps.
In nine subjects, one nasal polyp was removed surgically before treatment and
another was removed after 8 weeks of intranasal fluticasone (400 mg?day-1). Tissues
were processed for in situ hybridisation and immunohistochemical staining. Described
effects of fluticasone on nasal polyps (reduction in nasal resistance and in eosinophil
infiltration) were evaluated. Morphometric analysis was performed to assess the effect
of fluticasone on epithelial-, MUC5AC-, EGFR- and IL-8-stained areas, TNF-a-
stained cells, and neutrophil numbers.
Treatment with fluticasone decreased nasal resistance and intra-epithelial eosino-
phils. The MUC5AC-stained area in the epithelium was unchanged by treatment;
MUC5AC mRNA expression was unaffected by treatment. EGFR-stained area, intra-
epithelial neutrophil numbers, IL-8 and TNF-a expression were also unchanged by
Intranasal fluticasone was effective in decreasing nasal airflow resistance and intra-
epithelial eosinophils but had no effect on mucin or epidermal growth factor receptor
expression or on neutrophil recruitment.
Eur Respir J 2004; 24: 594–600.
*Service de Pneumologie, Universite ´ Rene ´
Descartes, Ho ˆpital Cochin, Paris, France.
#Dept of Otorhinolaryngology, Malmo ¨ Uni-
versity Hospital, Malmo ¨, Sweden.
vascular Research Institute, and Dept of
Medicine and Physiology, University of Cali-
fornia, San Francisco, CA, USA.
Correspondence: J.A. Nadel, Cardiovascular
Research Institute, Box 0130, University of
California San Francisco, San Francisco, CA
94 143-0130, USA.
Fax: 01 4154762283
Keywords: Airway epithelium
epidermal growth factor receptor
Received: February 4 2004
Accepted after revision: May 4 2004
This work was funded by private funds. P-R.
Burgel is a recipient of a grant from "Colle `ge
des Professeurs de Pneumologie".
Goblet cell hyperplasia and increased mucin expression
are features of nasal polyp epithelium , but, currently, no
effective therapy for mucus hypersecretion is established.
Eosinophil infiltration is a characteristic finding in nasal
polyp tissue. Neutrophils are recruited into the airways in
hypersecretory airway diseases (e.g. chronic obstructive
pulmonary disease, cystic fibrosis, bronchiectasis, acute asthma)
and are also found in nasal polyps . Neutrophil chemoat-
tractants are upregulated in the airways of these diseases 
and expression of the neutrophil chemoattractant interleukin
(IL)-8 is reported in nasal polyp tissue . Several neutrophil
products are reported to play important roles in mucin
production, including oxygen free radicals  and elastase .
Eosinophil products also increase mucin production in airway
epithelial cells . Therefore, both eosinophils and neutro-
phils have been suspected to play roles in the increased mucin
expression found in nasal polyps .
Corticosteroids are the recommended medical therapy
for nasal polyps. They are effective in decreasing the size of
polyps [8, 9] and in inhibiting eosinophil infiltration into
polyp tissue , but their effect on mucin production is not
Several mucins are expressed in human airways. Among
these mucins, gel-forming mucins found in airway secretions
include MUC2, MUC5AC and MUC5B. MUC2 expression
has not been found consistently in immunohistochemical
studies  and only a small amount of MUC2 protein has
been found in airway secretions . Although MUC5B is
reported to be expressed in goblet cells in normal nasal
epithelium, it is believed to be localised preferentially to
airway submucosal glands . Furthermore, there appear to
be no reports of MUC5B protein expression in nasal polyps.
In contrast, MUC5AC is consistently reported to be expres-
sed in both nasal and lower airway epithelium [1, 11]. We
have previously shown that MUC5AC gene and protein
are expressed in nasal polyp epithelium, and that MUC5AC-
stained area in nasal polyp epithelium is comparable to AB/
PAS-stained area . Because both epidermal growth factor
receptor (EGFR) activation  and leukocyte products have
been implicated in mucin production, the effects of intranasal
fluticasone on mucin MUC5AC and EGFR expression, and
on leukocyte infiltration were examined in this study.
In each subject, one polyp was removed before cortico-
steroid therapy and a second polyp was removed after 8 weeks
of treatment with an intranasal corticosteroid, fluticasone.
The effectiveness of fluticasone on the size of polyps (evalu-
ated by nasal airflow resistance) and eosinophil numbers was
assessed, both of which are known to be decreased by flutica-
sone . To assess the effect of fluticasone on mucin produc-
tion and its effect on the EGFR cascade, in situ hybridisation
and immunohistochemistry for MUC5AC gene, and protein
expression and immunohistochemistry for EGFR protein
Eur Respir J 2004; 24: 594–600
Printed in UK – all rights reserved
Copyright#ERS Journals Ltd 2004
European Respiratory Journal
were performed. Neutrophil infiltration, and expression of IL-8
and tumour necrosis factor (TNF)-a were also examined.
Nasal polyps were removed from nine subjects with bila-
teral nasal polyposis requiring surgical intervention. Nasal
polyposis was identified based on clinical symptoms (nasal
obstruction, anosmia, sneezing, excessive secretions, itching) and
based on the visualisation of polyps by anterior rhinoscopy.
Individuals with the following were excluded: aspirin-induced
airway symptoms, cystic fibrosis, ciliary dyskinesia, a history
of concurrent purulent nasal infection during the 6 weeks
before the study and nasal surgery during the past year. No
subjects suffered from asthma that required continuous
medication. Skin-prick tests were performed with a standard
panel of 10 common airborne allergens (ALK, Copenhagen,
Denmark) including pollen, house dust mites, mould and
animal allergens. Six out of nine subjects had a positive skin
test for at least one allergen. Although this study was not
designed to examine differences between atopic and non-
atopic subjects, no differences were observed between the two
groups in neutrophil or eosinophil infiltration, as described
previously . EGFR and MUC5AC mucin expression were
also similar in atopic and nonatopic subjects. Steroids were
withheld for a minimum of 6 weeks prior to the study. After
this run-in period, a polyp was removed surgically in each
subject; no other surgical procedures were performed. Two
weeks later, rhinomanometry was performed to measure nasal
airflow resistance, and then therapy with intranasal flutica-
sone (200 mg twice daily) was initiated. This regimen was
chosen based on the results of previous placebo-controlled
studies showing positive effects of fluticasone on polyp
symptoms, size and eosinophil infiltration [8, 10, 16]. Because
these effects of steroids on polyps are well described, they
were used as positive controls for efficacy of corticosteroid
delivery. Therefore, as reported in a previous study , a
placebo treatment group was not included. After 8 weeks of
fluticasone therapy, measurement of nasal airflow resistance
was repeated, and then another polyp was removed.
All studies were approved by the Ethics Committee of the
University of Malmo ¨ (Malmo ¨, Sweden) and an informed
written consent was obtained from each subject.
Nasal airflow resistance was determined for each nostril
using active anterior rhinomanometry , a method where
nasal airflow and pressure are measured at the nostrils during
respiration. Before performing rhinomanometry, subjects
rested in the sitting position. A pressure catheter was inserted
in a nostril that was occluded with a strip of adhesive tape,
and the other end was connected to a pressure transducer.
Care was taken to insure that the sides of the catheter did not
contact any part of the nasal mucosa. Flow was measured at a
pressure of 1.5 cmH2O with a pneumotachograph, via a mask
held airtight over the nose and mouth by the subject with as
little compression as possible. Nasal airflow resistance was
expressed in cmH2O?L-1?s-1.
Tissue processing and immunohistochemistry
Nasal polyps were removed surgically, fixed immediately in
10% formalin, embedded in paraffin and cut into 5-mm
sections. The sections were deparaffinised, rehydrated and
treated with 0.3% H2O2in methyl alcohol. PBS containing
0.05% Tween-20 and 1% bovine serum albumin (BSA) was
used as diluent for the following antibodies: a monoclonal
antibody (mAb) to MUC5AC (clone 45M1; dilution 1:500;
Neomarkers, Fremont, CA, USA); a mAb to EGFR (Ab-1;
dilution 1:100; Calbiochem, La Jolla, CA, USA); a mAb to
eosinophil cationic protein (EG2; dilution 1:1,000; Pharmacia
Upjohn, Kalamazoo, MI, USA); a mAb to human neutrophil
elastase (NP57; dilution 1:100; DAKO Corp., Carpinteria,
CA, USA); a polyclonal Ab to IL-8 (dilution 1:20; Biosource,
Camarillo, CA, USA); and a polyclonal rabbit anti-human
antibody to TNF-a (dilution 1:1,000; Genzyme Corp, Cam-
bridge, MA, USA).
Tissue sections were incubated with 2% BSA at room
temperature for 1 h and were then incubated with primary
antibody at room temperature for 2 h. After removing excess
Ab by washing with PBS, sections were incubated with a
biotinylated horse anti-mouse Ab (dilution 1:200; Vector
Laboratories, Burlingame, CA, USA) or with a biotinylated
goat anti-rabbit Ab (dilution 1:200; Vector Laboratories) for
1 h at room temperature. Bound Ab was visualised according
to standard protocols for avidin-biotin-peroxidase complex
method (Elite ABC kit; Vector Laboratories). Tissue sections
were counterstained with haematoxylin. Omission of the
primary Ab and incubation with an irrelevant mouse immuno-
globulin (Ig)G antibody (DAKO) were used as negative
controls. Alcian Blue (AB)/PAS was performed for detection
of mucous glycoconjugates.
In situ hybridisation for MUC5AC mRNA
MUC5AC gene expression was assessed by in situ
the probes and in situ hybridisation were performed as descri-
bed previously .
35S-labelled riboprobes. Preparation of
Eosinophil and neutrophil numbers
Quantification of eosinophil and neutrophil numbers in
polyp tissues was performed by counting numbers of human
neutrophil elastase (HNE)-stained (for neutrophils) and EG2-
stained (for eosinophils) cells in epithelium and subepithe-
lium. Epithelium was defined as the area between cilia and
basal membrane. Subepithelium was defined as the area
immediately underneath the basal membrane (depth: one high
power field at 4006 =0.0125 mm). For each layer (epithelium,
subepithelium), 10 high-power fields (magnification 4006)
were selected randomly and positively stained cells were
counted. Results are expressed as the number of cells per mm
of basal membrane in epithelium and as the number of cells
per mm2in subepithelium.
Epithelial, and MUC5AC- and EGFR-stained areas
Measurements of epithelial, and MUC5AC- and EGFR-
stained areas in polyp epithelium were performed using a
semi-automatic imaging system that included a microscope, a
video camera and a computer. Because of the heterogeneity
of polyp epithelium , for EGFR- and MUC5AC-stained
areas, and total epithelial area, data were obtained from two
first images of all intact epithelium were recorded at 1006
magnification. Next, epithelial area was measured on a compu-
ter screen using a grid composed of points; intersections of
points with epithelium were counted and converted to areas
(each point corresponding to a known area) . Results
obtained in two sections were averaged and expressed as mm2
of epithelium per mm of basal membrane. The median length
of epithelium analysed in each section was 15.1 mm (range
4.4–23.3). Areas of epithelium occupied by MUC5AC- and by
EGFR-stained cells were also measured by point counting
using a grid (supra vide). Results obtained in two sections
were averaged and were expressed as mm2of MUC5AC or
EGFR staining per mm of basal membrane. Results were not
different when they were expressed as % positively stained
area of epithelium.
Visualisation of staining for IL-8, a small diffusible
molecule, required higher magnification than visualisation
of MUC5AC and EGFR staining. Therefore, measurements
were performed at 4006magnification: ten images of epithe-
lium were randomly obtained, and areas of epithelium occu-
pied by IL8-stained cells were measured by point counting
using a grid (supra vide). Results were expressed as mm2of
IL-8 staining per mm of basal membrane.
As TNF-a in epithelium was expressed mostly in recruited
this molecule was assessed by counting positively stained cells
(both epithelial and inflammatory cells); for each polyp, 10
high-power fields (magnification 4006) were selected randomly
in a single section and positively stained cells were counted.
Results are expressed as cells per mm of basal membrane.
Data obtained from measurements of nasal airflow
resistance and from morphometric measurements of epithelial
area, MUC5AC-, EGFR- and IL-8-stained areas, and HNE-,
EG2- and TNF-a-stained cells before and after steroid
treatment were compared using the nonparametric Wilcoxon
signed-rank test. A p-valuev0.05 for the null hypothesis was
accepted as indicating a statistically significant difference.
Sections were evaluated by a single observer on two different
occasions in coded random order. All intra-observer coeffi-
cients of variation for repeated measurements werev9%.
Nasal airflow resistance
Patients with nasal polyps had variable nasal airflow resis-
tance before therapy. Intranasal fluticasone (400 mg, daily for
8 weeks)decreased nasal airflow resistance significantly (pv0.05,
Eosinophil and neutrophil numbers
Before therapy, eosinophils and neutrophils were present
in polyp epithelium. Fluticasone decreased EG2-positive
eosinophil numbers (pv0.01), but HNE-positive neutrophil
numbers were unaffected (p=0.84; fig. 2).
In the subepithelium of polyps, EG2-positive eosinophil
Fig. 1.–Nasal airflow resistance was measured by active anterior
rhinomanometry in subjects before (B) and after (A) treatment with
intranasal fluticasone. Fluticasone decreased airflow resistance (*:
pv0.05 compared to before fluticasone). Connecting lines indicate
data obtained in the same subject. Horizontal bars represent median
? ? ? ?
Fig. 2.–a) Eosinophils (assessed by EG2-stained cells) and b)
neutrophils (assessed by human neutrophil elastase (HNE)-stained
cells) were counted in polyp epithelium of subjects before (B) and
after (A) fluticasone treatment. Fluticasone decreased intra-epithelial
eosinophil (**: pv0.01) but not intra-epithelial neutrophil numbers
significantly (p=0.84). Data are presented as numbers of cells per mm
of basal membrane (BM). Connecting lines indicate data obtained in
the same subject. Horizontal bars represent median values. Vertical
bars indicate SEM.
P-R. BURGEL ET AL.
numbers were also decreased by fluticasone (median 28
cells?mm-2(range 2–88) before treatment versus 6 cells?mm-2
(0–42) after treatment, pv0.01), whereas HNE-positive neutro-
phil numbers wereunaffected
before treatment versus 17 cells?mm-2(6–70) after treatment,
Morphometric analysis of epithelial area and MUC5AC
and EGFR expression
The total epithelial area was slightly decreased after
treatment with fluticasone, although this did not reach
statistical significance (p=0.054; fig. 3a). As described pre-
viously , AB/PAS- and MUC5AC-stained areas were not
different from one another in polyp epithelium (data not
shown). MUC5AC staining was present in epithelial goblet
cells, and the MUC5AC-stained area was unaffected by
fluticasone (p=0.91) (fig. 3b). MUC5AC mRNA assessed by
in situ hybridisation showed strong signals before and after
steroid treatment (fig. 4). Hybridisation with a sense probe
for MUC5AC mRNA showed no signal (data not shown).
EGFR-positive staining was present in all polyp specimens
before and after treatment with fluticasone. Staining was
found especially in basal cells but also in Clara cells (fig. 4).
No staining for EGFR protein was found in mature mucin-
containing (goblet) cells. Fluticasone had no significant effect
on EGFR-stained area (p=0.82; fig. 3c). When antibodies
were omitted or when the staining procedure was performed
with an irrelevant mouse Ab, no staining was observed (data
Staining for IL-8 was present in nasal polyp epithelium and
was localised to basal and ciliated epithelial cells; however,
IL-8 was not present in goblet cells. Some inflammatory cells
infiltrating airway epithelium also expressed positive staining
for IL-8. IL-8-stained area in epithelium was not affected by
treatment with fluticasone (p=0.65; fig. 5a). No significant
correlation was found between intra-epithelial neutrophils
and IL-8 expression (r=-0.051, p=0.84, n=18 polyps, Spearman
TNF-a-expressing cells were regularly found in polyp
epithelium, mostly in inflammatory cells infiltrating polyp
epithelium; epithelial cells also expressed TNF-a. TNF-a
positively stained cell numbers were not different before and
after fluticasone treatment (p=0.82; fig. 5b).
Corticosteroids are useful in therapy of nasal polyps, but
their effects on mucin expression are not definitely estab-
lished. In the present study, mucin synthesis was unaffected
by corticosteroids. The lack of corticosteroid effect on mucin
production could not be due to ineffective drug delivery,
because the therapy decreased the polyp size (as manifested
by a decrease in nasal airflow resistance) and decreased
eosinophil infiltration, indicating that drug deposition was
effective. The present study was designed with these positive
controls, based on previous studies, which showed that reduc-
tion in polyp size and eosinophil infiltration are consistent
findings with intranasal corticosteroid therapy [8–10, 17, 20].
Evaluation of the effects of corticosteroids on mucins in
subjects with nasal polyps has not been reported, to our
knowledge. In lower airway epithelium of asthmatic patients,
available studies show conflicting data regarding effect of
local steroids on goblet cell metaplasia and mucin expression.
LAITINEN et al.  studied bronchial biopsies obtained before
treatment and after 3 months of treatment with budesonide
(600 mg, twice daily) in seven asthmatic subjects. The authors
reported significant increase in ciliated-to-goblet cell index
after treatment with inhaled budesonide. However, no data
were presented concerning mucin gene or protein expression.
GRONEBERG et al.  reported no change in mucin content
after a month of inhaled budesonide therapy, using a semi-
quantitative analysis of MUC5AC mucin staining of bronchial
biopsies. FAHY et al.  reported that a month of inhaled
beclomethasone therapy did not affect mucin-like glycopro-
teins in the sputum of moderate asthmatics. The present study
is the first to assess effects of local steroids in human airway
epithelium in vivo on both mucin gene and protein expression,
using quantitative morphometric analysis. The data presented
here show that mucin protein content in epithelium is
Fig. 3.–Data obtained from morphometric analysis of tissue sections stained with MUC5AC or epidermal growth factor receptor (EGFR)
monoclonal antibodies in subjects before (B) and after (A) fluticasone treatment. Areas, measured by point-counting, were expressed in mm2per
mm of basal membrane (BM). A small, insignificant decrease in a) epithelial area was found after therapy (p=0.054); no changes were found for
b) MUC5AC- or c) EGFR-stained areas (p=0.91 and p=0.82, respectively). Connecting lines indicate data obtained in the same subject.
Horizontal bars represent median values. Vertical bars indicate SEM.
unchanged by usual doses of intranasal steroids. The presence
of mucus in airway epithelium is related to mucin synthesis
(that increases mucin contents) and to mucin secretion (that
decreases mucin contents). Therefore, the persistence of mucin
protein expression in airway epithelium after treatment with
intranasal steroids may be related to effects on mucin synthesis
and/or mucin secretion. Importantly, using in situ hybridisa-
tion, it was found that MUC5AC gene expression is not
abolished by therapy. This study design did not allow for
studying mucin secretion. However, MACGREGOR et al. ,
measuring secreted mucous glycoproteins in nasal lavage fluid
in normal subjects, concluded that intranasal steroids potenti-
ate mucin secretion. Taken together, these results suggest that
local steroids do not inhibit either mucin synthesis or mucin
secretion and that alternative treatment is required to treat
No data are available regarding the magnitude of the
reduction in mucin content that is clinically significant, so the
number of subjects necessary to draw negative conclusions
could not be determined. Therefore, the possibility that intra-
nasal steroids have small effects on mucin expression cannot
be ruled out and studies analysing larger numbers of subjects
may show some positive effects.
A small decrease in total epithelial area was found after
steroid treatment. Because intranasal steroids had no effect
on mucin-producing cells, this finding could be related to
effects of steroids on other epithelial cell types (e.g. ciliated
cells, basal cells).
Fig. 4.–Representative photomicrographs of in situ hybridisation for MUC5AC mRNA (a and b), and immunolocalisation for MUC5AC
protein (c and d) and epidermal growth factor receptor (EGFR) protein (e and f), before (a, c and e) and after (b, d, and f) steroid treatment.
Before treatment, the in situ hybridisation signal for MUC5AC mRNA (seen as white dots under dark field) was strong in polyp epithelium;
MUC5AC protein was localised to epithelial goblet cells (arrows). Staining for EGFR protein was present in basal and Clara cells, but not in
mature mucus-containing (goblet) cells. After treatment with intranasal fluticasone no changes were observed in signals for MUC5AC mRNA,
MUC5AC protein or EGFR protein. Images are representative of findings in nine subjects for immunolocalisation and in four subjects for
in situ hybridisation. Scale bar=50 mm.
P-R. BURGEL ET AL.