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The Effect of Growth Factors on the Proliferation and Differentiation of Human Nasal Gland Cells
Tetsuro Kimura, MD;
Yuichi Majima, MD;
Yongqing Guo, MD;
Toshimichi Yoshida, MD
Arch Otolaryngol Head Neck Surg. 2002;128:578-582.
ABSTRACT
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Objective To elucidate a mechanism of proliferation and differentiation of nasal
gland cells, we established a serum-free 3-dimensional culture system for
human nasal gland (HNG) cells and examined the effects of epidermal growth
factor, keratinocyte growth factor, and retinoic acid on proliferation and
differentiation of cultured HNG cells.
Materials and Methods Nasal polyps were obtained from patients undergoing endoscopic endonasal
sinus surgery. The HNG cells were cultured under a monolayer culture and transferred
to a collagen-embedded culture using RPMI 1640 medium containing transferrin,
insulin, hydrocortisone, retinoic acid, epidermal growth factor, and keratinocyte
growth factor. Cell growth was measured by bromodeoxyuridine incorporation
assays. To measure cell differentiation, the percentage of cells containing
secretory granules, which were stained with Alcian blue in cytoplasm, was
determined.
Results In the serum-free 3-dimensional culture, the HNG cells showed ductal
structures containing secretory products in a lumen. The addition of epidermal
growth factor promoted the proliferation of HNG cells in its optimal concentrations,
and keratinocyte growth factor also enhanced the proliferation of HNG cells.
Conversely, the differentiation of HNG cells was not dependent on epidermal
growth factor and keratinocyte growth factor. Retinoic acid suppressed the
proliferation, but promoted the differentiation of HNG cells.
Conclusion Our culture system could be useful for studying the effects of various
growth factors and cytokines on HNG proliferation and differentiation to better
understand the mechanisms of growth and morphogenesis of nasal glands.
INTRODUCTION
SUBMUCOSAL GLANDS of respiratory tracts develop during organogenesis
and remodels in various pathological conditions. Especially in chronic inflammations,
glandular proliferation is remarkable. Mucous gland hyperplasia and hypertrophy
was reported in the tracheobronchial mucosa in chronic bronchitis.1 Nasal and sinus gland hyperplasia is a common feature
of chronic sinusitis.2-3 Such
glandular hyperplasia is suggested to be a main cause of viscous nasal hypersecretion.2 The hyperplasia is speculated to be regulated by some
growth factors and cytokines: however, there is little evidence about the
factors related to proliferation and differentiation of nasal subglandular
epithelia.
Our previous study described a 3-dimensional (3-D) culture system for
human nasal gland (HNG) cells. The HNG cells in this culture system promoted
glandular structures and secretory activity similar to the in situ conditions.4
To elucidate a mechanism of nasal gland hyperplasia, in the present
study we established a serum-free 3-D culture system for HNG cells. Using
this system, we examined the effects of growth factors and retinoic acid (RA)
on proliferation and differentiation of cultured HNG cells.
MATERIALS AND METHODS
CELL CULTURE
Nasal polyps were obtained from patients undergoing endoscopic endonasal
sinus surgery. The isolation of HNG cells was performed as described by Furukawa
et al.5 Nasal polyps were exposed overnight
at 4°C to 0.05% protease XIV (Sigma Chemical Co, St Louis, Mo) dissolved
in phosphate-buffered saline solution. The enzyme activity was stopped by
the addition of fetal calf serum to a final concentration of 2.5%, and small
sheets of the surface epithelial cells were dislodged from the nasal polyps
by vigorous agitation. The denuded nasal polyps with rich glands were collected
and rinsed 3 times with phosphate-buffered saline solution. The polyps were
minced and placed in Hanks solution containing 20mM HEPES, 500-U/mL collagenase
type IV, 6-U/mL elastase, 200-U/mL hyaluronidase, and 10-U/mL DNase (Sigma
Chemical Co) at room temperature for 4 hours. After the solution settled and
supernatant decanted, the isolated glands in the solution were collected by
centrifugation and resuspended in a mixture of RPMI 1640 medium (Sigma Chemical
Co) supplemented with 20% fetal calf serum. The glandular epithelia were plated
into a 25-cm2 tissue culture flask (T25) and incubated at 37°C
in 5% carbon monoxide and 95% air. The next morning, the medium was replaced
with RPMI 1640 medium containing 1-µg/mL transferrin, 1-µg/mL
insulin, 0.5-µg/mL hydrocortisone, 10-ng/mL RA(Sigma Chemical Co), and
10-ng/mL epidermal growth factor (EGF) (Becton, Dickinson and Company, Bedford,
Mass) as the basal medium. One week after cell plating, the confluent cells
were trypsinized in 0.05% trypsin and 0.02% EDTA solution and then collected
by centrifugation to transfer to the 3-D culture.
The conditioned medium used for the 3-D culture was RPMI 1640 medium
containing 1-µg/mL transferrin; 1-µg/mL insulin; 0.5-µg/mL
hydrocortisone; 0-, 30-, 100-, or 300-ng/mL RA; 0-, 3-,10-, or 30-ng/mL EGF;
and 0- or 15-ng/mL keratinocyte growth factor (KGF) (Strathmann Biotech GMBH,
Hannover, Germany). The collagen gels were prepared by mixing 8 mL of type
I collagen solution (3.0 mg/mL) (Nitta Gelatin Inc, Tokyo, Japan), 1 mL of
conditioned medium (concentration x10), and 1 mL of reconstitution buffer.
For the collagen-embedded system, 1 mL of this mixture (kept on ice) was first
placed in a 12-well plastic plate and immediately warmed to 37°C for gel
formation. One milliliter of the basal medium containing 1 x 105 cells was placed on collagen gel of a well. The cells were allowed
to attach to the surface of the gel for 12 hours. The medium and unattached
cells were removed, and a second layer of collagen was allowed to polymerize
on top of the first gel, thus embedding the cells. The gel was further covered
with 2 mL of the medium 30 minutes later. Three days after seeding the cells,
the gels were removed from plastic substratum by circling a spatula along
the inner edge of the well. While being cultured, the HNG cells were observed
under phase contrast microscope.
HISTOCHEMICAL STUDIES AND ELECTRON MICROSCOPY
After 7 days, the cells in the collagen gel were fixed with 10% formalin
in 0.1M phosphate buffer (pH 7.4) and routinely embedded in paraffin for histochemical
studies. Sections were stained with Alcian blue (pH 2.5), and nuclei were
counterstained with nuclear fast red. The percentage of cells containing secretory
granules stained with Alcian blue in cytoplasm were obtained to examine the
degree of cell differentiation (No. of mucus-producing cells–total No.
of cells [M/T ratio]). More than 200 cells were counted, and the given values
are the means of determinations from 3 individual experiments.
Cell growth fraction was determined by bromodeoxyuridine (BrdU) incorporation
assays.6 After 7 days, the cells in the collagen
gel were labeled with 10 µg/mL of BrdU (Sigma Chemical Co) for 4 hours
and then fixed with 10% formalin in 0.1M phosphate buffer (pH 7.4). Labeled
nuclei were detected with monoclonal anti-BrdU antibody (DAKO A/S, Copenhagen,
Denmark) and secondary anti-mouse IgG antibody conjugated with peroxidase
(Medical & Biological Laboratories Co Ltd, Nagoya, Japan), followed by
color development in diaminobenzidine hydrochloride solution. More than 200
cells were counted, and the percentage of nuclei that were labeled with BrdU
(labeling index) was determined. The given values of the indexes are the means
of determinations from 3 individual experiments.
For electron microscopic observations, the cells in the collagen gel
were fixed with 2.5% glutaraldehyde in 0.1M phosphate-buffered saline solution
at 4°C for 4 hours, followed by postfixation in 1% osmium tetroxide for
2 hours. The ultrathin sections were stained with 2% uranyl acetate and lead
citrate and observed using a transmission electron microscope (model H-800;
Hitachi, Tokyo, Japan).
RESULTS
MORPHOGENESIS OF 3-D–CULTURED HNG CELLS
After 7 days of 3-D culturing, the HNG cells in the conditioned medium
containing 10-ng/mL EGF and 100-ng/mL RA developed projected ductal structures
forming networks of the tubules. The cross-section shows a lumen containing
secretory products (Figure 1).
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Figure 1. Human nasal gland (HNG) cells
cultured in collagen gel (C) with serum-free culture medium. The sections
of cultured HNG cells form a lumen (L) containing mucus stained with Alcian
blue. The HNG cells were cultured for 7 days in the conditioned medium containing
10-ng/mL epidermal growth factor and 100-ng/mL retinoic acid. Sections were
stained with Alcian blue (pH 2.5), and nuclei were counterstained with hematoxylin-eosin
(original magnification x200). S indicates secretory products.
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Findings from electron microscopy revealed that 3-D–cultured HNG
cells have characteristics similar to gland cells. They were well polarized
and interconnected by junctional complexes and also had extensive development
of microvilli along the luminal surface and numerous secretory granules in
the cytoplasm (Figure 2).
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Figure 2. Transmission electron micrograph
of human nasal gland cells after being cultured for 7 days in collagen gel
with serum-free culture medium containing 10-ng/mL epidermal growth factor
and 100-ng/mL retinoic acid. The lumen (L) is bordered by polarized secretory
epithelial cells exhibiting microvilli (M) and numerous secretory granules
(SG) located along the L border (original magnification x18 960).
JC indicates junctional complexes.
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EFFECT OF EGF, KGF, AND RA ON HNG PROLIFERATION
Figure 3A-B shows BrdU-incorporated
HNG cells cultured in the defined medium containing 100-ng/mL RA with and
without EGF. The BrdU-incorporated nuclei were more prevalent in the cells
cultured in the medium containing 10-ng/mL EGF (Figure 3B) compared with those in EGF-free medium (Figure 3A). Also, the labeling indexes of the HNG cells cultured
in the medium containing 3- and 10-ng/mL EGF were significantly higher than
those in the EGF-free medium (Figure 4A).
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Figure 3. Bromodeoxyuridine incorporation
in human nasal gland cells. The human nasal gland cells were cultured in conditioned
medium containing 100-ng/mL retinoic acid without epidermal growth factor
(A) and with 10-ng/mL epidermal growth factor (B). Brown color (arrows) indicates
bromodeoxyuridine-labeled nuclei (original magnification x200).
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Figure 4. Percentage of bromodeoxyuridine-labeled
nuclei (labeling index) in cultured human nasal gland (HNG) cells. A, The
effect of epidermal growth factor (EGF) on HNG proliferation for HNG cells
cultured in conditioned medium containing 100-ng/mL retinoic acid (RA) and
0-, 3-, 10, and 30-ng/mL EGF. B, The effect of keratinocyte growth factor
(KGF) on HNG proliferation for HNG cells cultured in conditioned medium containing
100-ng/mL RA with 0- and 10-ng/mL EGF and 0- and 15-ng/mL KGF. C, The effect
of RA on HNG proliferation for HNG cells cultured in conditioned medium containing
10-ng/mL EGF and 0-, 30-, 100-, and 300-ng/mL RA. Brackets indicate significantly
different at P<.05. Error bars indicate SD.
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Figure 4B shows the labeling
index with conditioned medium containing 100-ng/mL RA. The addition of 15-ng/mL
KGF significantly increased the labeling index compared with the medium without
either KGF or EGF. There were no significant differences in the labeling index
between 15-ng/mL KGF and 10-ng/mL EGF. Synergetic effects were not observed
when 15-ng/mL KGF and 10-ng/mL EGF were concomitantly added to the medium.
The labeling index of HNG cells in the medium containing 10-ng/mL EGF
and 0-, 30-, 100-, or 300-ng/mL RA is shown in Figure 4C. Retinoic acid significantly decreased the labeling index
in a dose-dependent manner.
EFFECT OF EGF, KGF, AND RA ON HNG DIFFERENTIATION
Figure 5A-B shows cross-sections
of HNG cells cultured in the medium containing 10-ng/mL EGF with 0- and 300-ng/mL
RA. Quantitative analyses demonstrate that the frequency of Alcian blue–positive
cells (M/T ratio) in the culture containing 300-ng/mL RA was significantly
higher than that in the RA-deficient medium (Figure 6C). Neither EGF nor KGF showed any significant changes in
the M/T ratio under the presence of 100-ng/mL RA (Figure 6A-B).
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Figure 5. Mucus production of cultured human
nasal gland cells. The human nasal gland cells were cultured in conditioned
medium containing 10-ng/mL epidermal growth factor without retinoic acid (A)
and with 300-ng/mL retinoic acid (B). The blue spots (arrows) indicate secretory
granules stained with Alcian blue (pH 2.5) (original magnification x200).
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Figure 6. The ratio of the number of mucus-producing
cells to the number of total cells (M/T) of human nasal gland (HNG) cells.
A, The effect of epidermal growth factor (EGF) on HNG differentiation for
HNG cells cultured in conditioned medium containing 100-ng/mL retinoic acid
(RA) and 0-, 3-, 10-, and 30-ng/mL EGF. B, The effect of keratinocyte growth
factor (KGF) on HNG differentiation for HNG cells cultured in conditioned
medium containing 100-ng/mL RA with 0- and 10-ng/mL EGF and 0- and 15-ng/mL
KGF. C, The effect of RA on HNG differentiation for HNG cells cultured in
conditioned medium containing 10-ng/mL EGF and 0-, 30-, 100, and 300-ng/mL
RA. Bracket indicates significantly different at P<.05.
Error bars indicate SD.
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COMMENT
Our previous study showed that HNG cells cultured in the 3-D collagen
gel initiated glandularlike morphogenesis and secretory activity.4 To evaluate intrinsic or extrinsic factors influencing
the differentiation and proliferation of HNG cells, we cultured HNG cells
in the collagen gel with serum-free–defined medium supplemented with
transferrin, insulin, hydrocortisone, RA, and EGF. The cells cultured in this
medium also developed ductal structures forming networks of the tubules. The
structure showed a lumen containing secretory products in the histological
section (Figure 1). The cells were
positive for Alcian blue, revealing differentiation to mucous cells. Ultrastructural
morphology confirmed that the cells form glands and/or ducts having a polarity
and junctional complexes (Figure 2).
The secretory glandules were also seen in the cytoplasm.
A well-known growth factor of epithelial cells, EGF is found in various
body and secreted fluid and is related to the proliferation of gland cells,
such as lacrimal, salivary, mammary, and prostate gland cells. The effects
of EGF on the proliferation of nasal gland cells are easily speculated, but
have not been analyzed. In the present study, the labeling index of BrdU in
HNG cells was increased, according to increased concentrations of EGF up to
10 ng/mL. At 30-ng/mL EGF, however, the labeling index was suppressed in the
EGF-free medium (Figure 4A). The
results suggest that EGF promotes the proliferation of HNG cells in its optimal
concentrations. An immunohistochemical study demonstrates that EGF and EGF
receptors on bronchial epithelia and glands were upregulated in the asthmatic
airway.7 Epidermal growth factor could play
an important role in the proliferation of glandular epithelia in chronic inflammatory
diseases of the upper respiratory tracts, such as chronic sinusitis and nasal
allergy. In contrast, because EGF did not alter the M/T ratio (Figure 6A), differentiations of HNG cells are dependent on factors
other than EGF.
Keratinocyte growth factor is a member of fibroblast growth factor family.
It stimulates the proliferation of a broad range of epithelial cells including
skin, bronchus, gastrointestinal tract, and mammary gland.8
In the present study, the labeling index of BrdU was significantly increased
in the HNG cells cultured in the 15-ng/mL KGF medium compared with those in
the medium without growth factors (Figure
4B), indicating that KGF enhanced the proliferation of HNG cells.
Enhanced expression of KGF and KGF-receptor mRNA was reported in the nasal
mucosa of chronic sinusitis.9 Taken together,
KGF may contribute to nasal and sinus gland hyperplasia observed in chronic
sinusitis.2 Because KGF has a synergetic effect
with mammogenic hormones on the proliferation of mammary gland cells,10 we examined an effect of KGF in combination with
EGF on the proliferation of HNG cells. However, no synergetic actions of KGF
and EGF was detected (Figure 4B). Keratinocyte growth factor enhances differentiation of alveolar type II cells
and/or their progenitors and promotes alveolarization during branching morphogenesis.9 In the present study, however, KGF, as well as combinations
of KGF and EGF, had no effect on the M/T ratio of HNG cells (Figure 6B) and may have little relation to the differentiation of
HNG cells.
Beta carotene and related compounds, summarily called retinoids, are
known to regulate cell proliferation, differentiation, and morphogenesis.
Depending on the cell or tissue type, retinoids induce or inhibit such regulation.11 Retinoic acid was reported to inhibit squamous differentiation
of epithelial cells and was essential for the maintenance of mucociliary epithelium.12 Also, RA up-regulated mucous gene expressions in
the retinoid-deficient cultures of airway epithelial cells.11
In the HNG cells, RA increased the M/T ratio in a dose-dependent manner (Figure 4C and Figure 6C), while it decreased the BrdU-labeling index. This clearly
indicates that RA promotes the differentiation but suppresses the proliferation
of the cells.
Hyperplasia of submucosal gland cells observed in chronic inflammations
of upper and lower respiratory tracts is investigated from an aspect of factors
influencing the proliferation and differentiation of the glandular cells.
Our 3-D culture system with a defined culture medium could be useful for better
understanding mechanisms of gland cell hyperplasia.
In conclusion, we established serum-free 3-D culture system for HNG
cells. In this condition, morphogenesis of HNG cells was similar to the in
situ condition. The differentiation and proliferation of HNG cells was probably
related, at least in part, to EGF, KGF, and RA. Our culture system is useful
to study effects of growth factors, cytokines, and various substances on the
proliferation and differentiation of HNG cells in morphogenesis during development
and remodeling under various pathological conditions.
AUTHOR INFORMATION
Accepted for publication October 2, 2001.
This report was supported by a grant-in-aid for general scientific research
(grant 10470355) from the Ministry of Education, Science, and Culture of Japan,
Tokyo.
Corresponding author and reprints: Yuichi Majima, MD, Department
of Otorhinolaryngology, Mie University School of Medicine, 2-174 Edobashi,
Tsu, Mie 514-8507, Japan.
From the Departments of Otorhinolaryngology (Drs Kimura, Majima, and
Guo) and Pathology (Dr Yoshida), Mie University School of Medicine, Tsu, Mie,
Japan.
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