Retinoic Acid Inhibits Elastase-Induced Injury in Human Lung
Epithelial Cell Lines
Mizue Nakajoh, Takeyasu Fukushima, Tomoko Suzuki, Mutsuo Yamaya, Katsutoshi Nakayama,
Kiyohisa Sekizawa, and Hidetada Sasaki
Department of Geriatric and Respiratory Medicine, Tohoku University School of Medicine, Sendai; and
Department of Respiratory Medicine, Institute of Clinical Medicine, University of Tsukuba, Tsukuba, Japan
The protective effects of retinoic acid on elastase-induced lung
epithelial cell injury were studied using elastase extracted from
purulent human sputum, the BEAS-2B human bronchial epithe-
lial cell line, A549 human type II lung cell line, and primary
cultures of human tracheal epithelial cells. Elastase decreased
lial cells in concentration- and time-dependent fashions. Elas-
tase also induced apoptosis of BEAS-2B cells, A549 cells, and
the tracheal epithelial cells detected with cell death detection
enzyme-linked immunosorbent assay and terminal deoxyribo-
of BEAS-2B cells, A549 cells, or the tracheal epithelial cells, and
did not induce apoptosis of the cells. However, retinoic acid
prevented the decreases in the viability and reduced apoptosis
of BEAS-2B cells, A549 cells, and the tracheal epithelial cells
induced by elastase. Likewise, retinoic acid inhibited caspase 3
activity in BEAS-2B cells and A549 cells induced by elastase, as
well as proteolytic activity of elastase. Furthermore, caspase 3
inhibitor inhibited the elastase-induced apoptosis of the cells.
These findings suggest that retinoic acid may inhibit elastase-
induced lung epithelial cell injury partly through the inhibition
of proteolytic activity of elastase and through the inhibition of
caspase3 activityby elastase.Retinoic acidmay,therefore, have
protective effects against the elastase-induced lung injury and
subsequent development of pulmonary emphysema.
To understand the pathogenesis of pulmonary emphy-
sema, animal models of emphysema using elastase have
induces perivascular edema and an alveolar hemorrhage in
the lung at the initial stage. The elastic framework of the
lung is subsequently disrupted and the alveoli are enlarged
and distorted (9, 10). Because elastase damages the airway
epithelial cells and vascular endothelial cells (11–13), the
hemorrhagic lung injury after intratracheal elastase instilla-
tion (9, 10) may be, in part, associated with the subsequent
development of pulmonary emphysema, although damage
by elastase are major causes of pulmonary emphysema
(3–5). Furthermore, a recent report suggested the relation
between lung cell apoptosis and emphysema in rats (14).
Massaro and coworkers (15) recently reported that reti-
noic acid inhibits elastase-induced pulmonary emphysema
in rats. Retinoic acid has a variety of biological activities
in the growth and differentiation of epithelial cells and in
age in the fibroblast in the alveolar wall is related to the
formation of alveolar septa (19). Although retinoic acid
induces apoptosis of various cells (20, 21), it inhibits hydro-
gen peroxide–induced apoptosis in mesangeal cells (22).
However, the mechanisms of the inhibitory effects of reti-
noic acid on elastase-induced pulmonary injury and subse-
quent development of pulmonary emphysema in rats have
not been studied.
In the present study, we examined whether retinoic acid
inhibits the injury and apoptosis of the cells induced by
elastase, and studied the mechanisms responsible for the
lial cell line, and the primary cultures of human tracheal
epithelial (HTE) cells.
Chronic obstructive pulmonary disease, which includes
and chronic bronchitis (1), is one of the leading causes of
ity (2). Cigarette smoke is the most common identifiable
risk factor for chronic obstructive pulmonary disease, and
two current hypotheses, the endogenous protease/antipro-
tease theory (3–6) and the oxidant/antioxidant theory
(7, 8), have been established in the pathogenesis of chronic
Materials and Methods
Reagents for cell culture media were obtained as follows: Dulbec-
co’s modified Eagle’s medium (DMEM), Ham’s F-12 medium,
phosphate-buffered saline (PBS), and fetal calf serum (FCS) were
from GIBCO-BRL Life Technologies (Palo Alto, CA); trypsin,
EDTA, penicillin, streptomycin, gentamicin, cholera toxin, and
all trans retinoic acid were from Sigma (St. Louis, MO); insulin,
transferrin, epidermal growth factor, endothelial cell growth sup-
plement, hydrocortisone, and triiodothyronine were from Becton
Dickinson (Collaborative Research Brand; Franklin Lakes, NJ);
and elastase from purulent human sputum was from Elastin Prod-
ucts (Owensville, MO). Elastase solution used in the present study
(Received in original form February 19, 2002 and in revised form September
Address correspondence to: Hidetada Sasaki, M.D., Professor and Chair-
man, Department of Geriatric and Respiratory Medicine, Tohoku Univer-
sity School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai, 980-8574 Japan.
Abbreviations: Dulbecco’s modified Eagle’s medium, DMEM; dimethyl
sulfoxide, DMSO; enzyme-linked immunosorbent assay, ELISA; fetal calf
serum, FCS; human tracheal epithelial cells, HTE cells; phosphate-buffered
saline, PBS; terminal deoxyribonucleotidyl transferase-mediated dUTP-
biotin nick-end labeling, TUNEL.
Am. J. Respir. Cell Mol. Biol.Vol. 28, pp. 296–304, 2003
Internet address: www.atsjournals.org
Nakajoh, Fukushima, Suzuki, et al.: Retinoic Acid and Epithelial Cell Injury297
contained 875 U/mg protein. Elastase was purified from human
sputum by ion exchange and affinity chromatography methods
(personal communications from Elastin Products). Purity of the
elastase was greater than 95% by SDS-PAGE, and the elastase
did not contain elastase inhibitor or significant levels of endotoxin
(? 10 EU) measured with the E-Toxate Multiple Test (personal
communications from Elastin Products).
by centrifugation (200 ? g, 10 min) and stored at 4?C for the assay.
To bind histone-associated DNA oligonucleosomes in superna-
tants to a biotinylated antihistone antibody, samples of superna-
tants were incubated in microtiter plates coated with streptavidin
conjugated with biotinylated mouse antihistone antibody (clone
H11–4). Plates were washed, and nonspecific binding sites were
saturated with an anti-mouse DNA monoclonal antibody (MCA-
33) and then conjugated with an anti-DNA antibody bound to
peroxidase. To determine the amount of retained peroxidase, 2,2?-
azino-di (3-ethylbenzthiazoline-6-sulfonate) (ABTS) was added as
a substrate, and the complex was measured by spectrophotometer
(Labsystems MultiskanBICHROMATIC; Labsystems) at405 nm.
Results are expressed as the ratio of sample absorbance to ab-
sorbance of the room air control sample measured daily.
Assessment of DNA fragmentation associated with apoptosis
of BEAS-2B cells and A549 cells was also performed by TUNEL
assay with the MEBSTAIN ApoptosisKit (Medical and Biological
Laboratories, Nagoya, Japan) as previously described (26). Cells
were washed, fixed, permeabilized, and labeled with avidin-FITC
number of FITC-labeled cells was counted under a fluorescent
microscopy (Meridian Instruments, Okemos, MI).
Human Epithelial Cell Culture
The BEAS-2B cells were supplied by Dr. Cutis Harris of the
supplied by the Cell Resource Center for Biomedical Research,
Institute of Development, Aging and Cancer, Tohoku University.
The BEAS-2B cells (1 ? 106cells) were cultured in T25flasks
(Costar Corning, Cambridge, MA) in a serum-free medium con-
sisting of DMEM–Ham’s F-12 medium (50/50, vol/vol) and the
25 ng/ml of epidermal growth factor, 7.5 ?g/ml of endothelial cell
growth supplement, 20 ng/ml of triiodothyronine, 0.36 ?g/ml of
hydrocortisone, and20 ng/ml ofcholera toxin. The A549cells were
cultured in T25flasks in DMEM supplemented with 8% FCS. The
100 mg/liter of streptomycin, and 50 mg/liter of gentamicin. After
cells made confluent cell sheets, cells were collected by trypsiniza-
tion (0.05% trypsin and 0.02% EDTA), replaced in cell culture
medium and antibiotics in 96-well plates (4 ? 104/0.2 ml) and
cultured at 37?C in 5% CO2–95% air. When A549 cells made
confluent sheets in 96-well dishes, the cells were rinsed with PBS
and further cultured in 200 ?l of the DMEM–Ham’s F-12 with
The HTE cells were isolated and plated in 96-well dishes with
the methods as previously described (23), in 200 ?l of the DMEM–
Ham’s F-12with 2% UltroserG serum substitute(USG; BioSepra,
Assessment of Caspase 3 Activity
Assessment of caspase 3 activity was performed by fluorometric
immunosorbent enzyme assay (FIENA) according to the instruc-
tion manual of the Caspase 3 Activity Assay Kit (Roche, Mann-
heim, Germany). BEAS-2B cells (2 ? 106), cultured in 6-well
culture dishes, were collected with scrapers and suspended in 1 ml
of PBS in 15-ml centrifuge tubes (Corning). Pellets of the cells
gation (600 ? g, 5 min, 4?C), pellets of the cells were lysed in 200
?l of buffer containing 1? DTT, and incubated for 1 min at 4?C.
The supernatants of cell lysates were collected by centrifugation
at maximum speed in a table top centrifuge (13,000 rpm, 1 min)
and were stored at 4?C for the assay. Samples of supernatants were
incubated with microtiter plates absorbed with anti-caspase 3 (27).
Plates were washed, and nonspecific binding sites were saturated
with the blocking buffer of the Kit. Plates were conjugated with
fluorescence substrate solution containing 50 ?M of acetyl-Asp-Glu-
and incubated for 2 h at 37?C. Ac-DEVD-AFC is cleaved propor-
tionally to the amount of activated caspase 3 and generates free
fluorescent 7-amido-4-trifluoromethyl-coumarin (AFC). The con-
tent of free AFC was measured fluorometrically at 505 nm in
excitation at 400nm by a fluorescencereader (Fluoroskan; Labsys-
tems). The fluorescence intensity is expressed as raw fluorescence
intensity minus background fluorescence intensity.
Assessment of Cell Viability
Cell viability was assessed with colorimetric MTT (tetrazolium)
assay with a Cell Counting Kit (Dojindo, Kumamoto, Japan) (24).
BEAS-2B cells and A549 cells cultured in 200 ?l of the DMEM–
Ham’s F-12 with growth factors, and primary cultures of HTE cells
cultured in 200 ?l of the DMEM–Ham’s F-12 with 2% USG in
96-well dishes were treated with elastase in the presence or absence
of retinoic acid for 24 h. Cells were then treated with 10 ?l of tetra-
20 mM HEPES, and 0.2 mM 1-methoxyl-5-methylphenazinium
methylsulfate (1-methoxyl PMS). The cells in the dishes were then
incubated at 37?C for 1 h in 5% CO2–95% air. The absorbance
intensity of the solution was measured on a spectrophotometer
(Labsystems Multiskan BICHROMATIC; Labsystems, Helsinki,
Finland) using a test wavelength of 450–690 nm.
Proteolytic Activity of Elastase
Specific proteolytic activity of elastase was determined by me-
by the method of Fujimoto and coworkers (28) with some modifi-
To study the effects of either retinoic acid or ?1-antitrypsin
on the proteolytic activity of elastase, 0.1 ?l of retinoic acid, ?1-
antitrypsin, or the vehicle of inhibitors was added to the reaction
mixture (100 ?l) containing 75 ?l of HEPES-NaCl buffer (0.1 M
HEPES, 0.5 M NaCl, pH 7.5) and 25 ?l of methoxysuccinyl-Ala-
Ala-Pro-Val-p-nitroanilide (6 ?g/ml). Methoxysuccinyl-Ala-Ala-
Pro-Val-p-nitroanilide was dissolved in the HEPES-NaCl buffer
containing 30% dimethyl sulfoxide (DMSO). Elastase was diluted
Assessment of DNA Fragmentation by Cell Death
Detection Enzyme-Linked Immunosorbent Assay
Assessment of DNA fragmentation associated with apoptosis was
performed by enzyme-linked immunosorbent assay (ELISA) with
cell death detection ELISA (Boehringer Mannheim, Indianapolis,
IN) as previous described (25). Either BEAS-2B cells, A549 cells,
or HTE cells were treated with elastase in the presence or absence
of retinoic acid for 24 h. The culture supernatants were collected
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