Bleomycin stimulates lung epithelial cells to release
neutrophil and monocyte chemotactic activities
Etsuro
Sato1,
Sekiya
Koyama1,
Takeshi
Masubuchi1,
Akemi
Takamizawa1,
Keishi
Kubo1,
Sonoko
Nagai2, and
Takateru
Izumi2
1 The First Department of
Internal Medicine, Shinshu University, School of Medicine,
Matsumoto 390; and 2 Kyoto
University Chest Disease Research Institute, Kyoto 606-01, Japan
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ABSTRACT |
Although
bleomycin, an antineoplastic drug, is used in the treatment of a
variety of tumors, the mechanisms of bleomycin-induced lung injury and
fibrosis are not fully elucidated. We postulated that bleomycin might
stimulate A549 cells, a type II pneumocyte cell line, to release
neutrophil and monocyte chemotactic activities (NCA and MCA,
respectively). To test this hypothesis, A549 cell supernatant fluids
were harvested and evaluated for NCA and MCA. A549 cell supernatant
fluids showed NCA and MCA in response to bleomycin in a dose- and
time-dependent manner (P < 0.05).
Checkerboard analysis revealed that both NCA and MCA were predominantly
chemotactic. Partial characterization of the released NCA and MCA
showed that the activities were partially heat labile, trypsin
digested, and predominantly ethyl acetate extractable. Lipoxygenase
inhibitors and cycloheximide inhibited the release of chemotactic
activities significantly. Molecular-sieve column chromatography
revealed that the released activities were heterogeneous. However,
low-molecular-weight activity was prominent. Leukotriene
B4-receptor antagonist,
anti-interleukin-8, anti-granulocyte colony-stimulating factor, and
anti-monocyte chemoattractant protein-1 antibodies attenuated the
chemotactic activities. Immunoreactive leukotriene
B4 receptor, interleukin-8, granulocyte colony-stimulating factor, and monocyte chemoattractant protein-1 significantly increased in supernatant fluids in response to
bleomycin. These data demonstrate that bleomycin stimulates type II
epithelial cells to release chemotactic activities and plays a role in
inflammatory cell recruitment into the lung.
type II epithelial cell; interleukin-8; monocyte chemoattractant
protein-1; granulocyte colony-stimulating factor; leukotriene
B4
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INTRODUCTION |
BLEOMYCIN, AN ANTICANCER AGENT, sometimes causes
diffuse interstitial lung fibrosis in humans. Therefore, clinical usage
of bleomycin is limited by this life-threatening disease. The incidence of pulmonary fibrosis varies from 3 to 40% (1, 2, 12, 17, 31, 32) and
fatality is 1-15% (4, 6) of patients who received this agent.
Because bleomycin-induced pulmonary fibrosis in animals resembles human
idiopathic pulmonary fibrosis histopathologically, bleomycin has been
used to induce pulmonary fibrosis in laboratory animals (13, 21).
However, the mechanisms of pulmonary fibrosis induced by bleomycin
still remain to be elucidated. Especially the role of type II alveolar
epithelial cells is uncertain in bleomycin-induced pulmonary fibrosis.
Inflammatory cell recruitment, which causes the lung injury, is a
significant event in pulmonary fibrosis (9). In experimental models of
bleomycin-induced pulmonary fibrosis as well as in human pulmonary
fibrosis by therapeutically used bleomycin, the histological features
include inflammatory cell recruitment, fibroblast proliferation, and
collagen synthesis (7). Inflammatory cells, including neutrophils and
monocytes, have the capacity to release toxic oxygen metabolites, elastolytic enzymes, and cytokines that cause lung injury.
Although alveolar epithelial cells have previously been regarded as
passive bystanders in immune interactions, it is now recognized that
these cells play a role in regulating the lung immune environment. Type
II epithelial cells release monocyte chemoattractant activity constitutively and express interleukin (IL)-8, monocyte chemoattractant protein-1 (MCP-1), regulated on activation normal T cells expressed and
secreted (RANTES), granulocyte-macrophage colony-stimulating factor
(GM-CSF), and transforming growth factor (TGF)-
in response to tumor
necrosis factor (TNF)-
and IL-1 (15, 28, 29). These cytokines have
the potential to attract and activate inflammatory cells, leading to
the lung injury. Because inflammatory cell recruitment into the lungs
plays important roles in bleomycin-induced lung injury and because type
II alveolar epithelial cells participate in the lung inflammatory
responses, we hypothesized that type II epithelial cells may release
neutrophil (NCA) and monocyte (MCA) chemotactic activities in response
to bleomycin. The results demonstrate that a human type II alveolar
epithelial-like cell line (A549 cells) released NCA and MCA, including
IL-8, granulocyte colony-stimulating factor (G-CSF), MCP-1, and
leukotriene (LT) B4 as chemotactic
factors, in response to bleomycin.
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MATERIALS AND METHODS |
Preparation of A549 type II alveolar epithelial
cells. Because of the difficulty in obtaining primary
human type II epithelial cells of sufficient purity, A549 cells
(passage 75; American Type Culture
Collection, Manassas, VA), a pulmonary type II epithelial cell line
derived from an individual with alveolar cell carcinoma, were used
(16). These cells retained many of the characteristics of normal type
II epithelial cells such as surfactant production, cytoplasmic
multilamellar inclusion bodies, and cuboidal appearance (8, 28, 29).
A549 cells were grown as monolayers in 100-mm tissue culture dishes.
A549 cells were incubated in 100% humidity and 5%
CO2 at 37°C with Ham's F-12
medium supplemented with penicillin (50 U/ml), streptomycin (50 µg/ml), Fungizone (2 µg/ml), and 10% heat-inactivated fetal calf
serum (FCS; all from GIBCO BRL, Grand Island, NY). Cells from the
monolayers were harvested with trypsin (0.25%) and EDTA (0.1%) in
PBS, centrifuged at low speed (250 g
for 5 min), and resuspended in fresh medium at a concentration of 1.0 × 106 cells/ml in 35-mm
tissue culture dishes. The cells were grown to confluence on the dish
for 5-7 days. After the cells reached confluence, they were used
for the experiments.
Exposure of A549 cells to bleomycin.
Medium was removed from the cells by washing two times with serum-free
Ham's F-12 medium, and the cells were incubated in the presence and
absence of bleomycin. To determine the dose-dependent release of NCA
and MCA, the cultures were incubated for 12, 24, 48, and 72 h at
37°C in a humidified 5% CO2
atmosphere at various concentrations of bleomycin (0, 0.1, 1.0, and 10 µg/ml; Sigma, St. Louis, MO). Bleomycin did not cause A549 cell
injury (no deformity of cell shape, no detachment from tissue culture
dish, and >95% of cells were viable by trypan blue exclusion) after
72 h of incubation at the maximal doses. The supernatant fluids were
harvested and stored at
80°C until assayed. At least six
separate A549 cell supernatant fluids were harvested from the cultures
for each experimental condition.
Measurement of NCA and MCA.
Polymorphonuclear leukocytes were purified from heparinized normal
human blood with the method of Boyum (5). Briefly, 15 ml of venous
blood were obtained from healthy volunteers, then sedimented with 3%
dextran in isotonic saline for 45 min to separate the white blood cells
from the red blood cells. The leukocyte-rich upper layer was collected,
and the neutrophils were separated from the mononuclear cells by
Ficoll-Hypaque density centrifugation (Histopaque 1077, Sigma).
Contaminating red blood cells were removed with a lysing solution
containing 0.1% KHCO3 and 0.83%
NH4Cl. The suspension was then
centrifuged at 400 g for 5 min and
washed three times in Hanks' balanced salt solution. The resulting
cell pellet, as determined by trypan blue and erythrosin exclusion,
consisted of >96% neutrophils and >98% viable cells. The cells
were suspended in Gey's balanced salt solution (GIBCO BRL) containing
2% bovine serum albumin (BSA; Sigma) at pH 7.2 to give a final
concentration of 3.0 ×106
cells/ml. This suspension was used for the neutrophil chemotaxis assay.
Mononuclear cells for the chemotaxis assay were obtained from normal
human volunteers and were separated from the red blood cells and
neutrophils by Ficoll-Hypaque density centrifugation. The mononuclear
cells were harvested at the interface. The suspension was then
centrifuged at 400 g for 10 min and
washed three times in Hanks' balanced salt solution. The preparation
routinely consisted of 30% large monocytes and 70% small lymphocytes
as determined by morphology and
-naphthyl acetate esterase staining
(Sigma), with >98% viability as assessed by trypan blue and
erythrosin exclusion. The cells were suspended in Gey's balanced salt
solution containing 2% BSA at pH 7.2 to give a final concentration of
5.0 × 106 cells/ml. The
suspension was then used for the monocyte chemotaxis assay.
The chemotaxis assay was performed in a 48-well microchemotaxis chamber
(Neuro Probe, Cabin John, MD) as previously described (11). The bottom
wells of the chamber were filled with 25 µl of fluid containing the
chemotactic stimulus or medium in duplicate. A 10-µm-thick
polyvinylpyrrolidone-free polycarbonate filter with a pore size of 3 µm for neutrophil chemotaxis and 5 µm for monocyte chemotaxis was
placed over the bottom wells. The silicon gasket and upper pieces of
the chamber were applied, and 50 µl of the cell suspension were
placed in the upper wells above the filter. The chambers were incubated
in humidified air in 5% CO2 at
37°C for 30 min for neutrophils and 90 min for monocytes.
Nonmigrated cells were wiped away from the filter. The filter was then
immersed in methanol for 5 min, stained with Diff-Quik, and mounted on a glass slide. Cells that completely migrated through the filter were
counted with light microscopy in 10 random high-power fields (HPF;
×1,000) per well. To ensure that monocytes, but not lymphocytes, were the primary cells that migrated in the monocyte chemotaxis assays,
some membranes were stained with
-naphthyl acetate esterase according to the manufacturer's instructions (Sigma).
To determine whether the migration was due to movement along a
concentration gradient (chemotaxis) or stimulation of random migration
(chemokinesis), a checkerboard analysis was performed with A549 cell
supernatant fluids harvested after 72 h of incubation with 10 µg/ml
of bleomycin (34). To do this, various dilutions of A549 cell
supernatant fluids (1:1, 1:4, 1:16, 1:64, and 1:256) were placed below
the membrane and above the membrane with target cells. Thus neutrophil
and monocyte migrations were tested by a variety of concentration
gradients of supernatant fluids across the membrane.
Partial characterization of NCA and
MCA. Partial characterization of NCA and MCA released
from A549 cells was performed with supernatant fluids harvested after
72 h of incubation with bleomycin at a concentration level of 10 µg/ml. Sensitivity to proteases was tested by incubating the
supernatant fluids with trypsin (100 µg/ml; Sigma) for 30 min at
37°C followed by the addition of a 1.5 molar excess of soybean
trypsin inhibitor to terminate the proteolytic activity, and then the
chemotactic activity was evaluated. The lipid solubility was evaluated
by mixing the supernatant fluids two times with ethyl acetate,
decanting the lipid phase after each extraction, evaporating the ethyl
acetate to dryness, and resuspending the extracted material in the
Ham's F-12 medium used for the cell culture before the chemotaxis
assay. Both extracted and extractant materials were evaluated for
chemotactic activity. Heat sensitivity was determined by heating the
supernatant fluids at 98°C for 15 min.
Molecular-sieve column chromatographic findings of the
released chemotactic activity. To determine the
approximate molecular mass of the released chemotactic
activity in the supernatant fluids harvested after 72 h of incubation
with 10 µg/ml of bleomycin, molecular-sieve column chromatography was
performed with Sephadex G-200 (Pharmacia, Piscataway, NJ). At a flow
rate of 6 ml/h, A549 cell culture supernatant fluids were eluted with
PBS, and the fractions were evaluated for NCA and MCA in duplicate.
Effects of metabolic inhibitors on the release of
chemotactic activity. The effects of the nonspecific
lipoxygenase inhibitors nordihydroguaiaretic acid (NDGA; 10 µM;
Sigma) and diethylcarbamazine (DEC; 1 mM; Sigma) and the 5-lipoxygenase
inhibitor AA-861 (100 µM; Takeda Pharmaceutical, Tokyo, Japan) on the
release of NCA and MCA in A549 cell supernatant fluid harvested after
72 h of incubation with 10 µg/ml of bleomycin were evaluated. To
further examine the involvement of protein synthesis in the release of chemotactic activity, cycloheximide (20 µg/ml; Sigma) was added to
inhibit protein synthesis (10).
Effects of LTB4- and platelet-activating
factor-receptor antagonists on chemotactic activity.
Because the release of NCA and MCA was blocked by 5-lipoxygenase
inhibitors and because NCA and MCA were extracted into ethyl acetate,
an LTB4-receptor antagonist
(ONO-4057, ONO Pharmaceutical, Tokyo, Japan) and a platelet-activating
factor (PAF)-receptor antagonist (TCV-309, Takeda Pharmaceutical,
Tokyo, Japan) at a concentration of
10
5 M were used to evaluate
the involvement of LTB4 and PAF as
NCA and MCA (14, 30). These receptor antagonists at a concentration of
10
5 M completely blocked
the neutrophil and monocyte chemotactic responses to
10
7 M
LTB4 and PAF. But these receptor
antagonists did not have any influence on the chemotactic response of
neutrophils and monocytes to endotoxin-activated serum and
N-formyl-methionyl-leucyl-phenylalanine (fMLP).
Measurement of LTB4 and PAF in the
supernatant fluid.
The concentration of LTB4 in the
supernatants was measured by radioimmunoassay (RIA) as previously
described (22, 23, 26). Anti-LTB4
serum,
[5,6,8,9,11,12,14,15-3H(N)]-LTB4,
and synthetic LTB4 were purchased
from Amersham (Arlington Heights, IL). Briefly, ethanol and the
supernatant mixtures were centrifuged at 5,500 g at 0°C. At a temperature of
37°C, the supernatants were evaporated under
N2 gas to remove the ethanol. To
each sample, 10 ml of distilled water were added. These samples were
acidified to pH 4.0 with 0.1 M hydrochloric acid and applied to Sep-Pak C18 columns (Waters Associates,
Milford, MA). The columns were washed with 10 ml of distilled water and
20 ml of petroleum ether, then eluted with 15 ml of methanol. These
eluates were dried with N2 gas at
37°C, then redissolved in 20 µl of methanol and 180 µl of RIA
buffer [50 mM Tris · HCl buffer containing
0.1% (wt/vol) gelatine, pH 8.6].
[3H]LTB4
was diluted in RIA buffer (0.1 ml containing ~4,000 dpm) and mixed
with 0.1 ml of standards or samples in disposable siliconized tubes.
Anti-LTB4 serum diluted with RIA
buffer (0.1 ml) was added to the siliconized tubes to give a total
incubation volume of 0.4 ml. The mixture was incubated at 4°C for
18 h. Free LTB4 was absorbed onto
dextran-coated charcoal. The supernatant containing the antibody-bound
LTB4 was decanted into a
scintillation counter vial after centrifugation for 15 min at 2,000 g. Scintillation fluid (Aquazol 2, NEN, Boston, MA) was added, and radioactivity was counted by a
scintillation counter (Tricarb-3255, Packard) for 4 min.
PAF in the supernatant fluids was evaluated via the scintillation
proximity assay system. Briefly, this assay system combined the use of
a high specific activity tritiated PAF tracer with an antibody specific
for PAF and a PAF standard similar to the methods for the measurement
of LTB4.
Effects of polyclonal antibodies to IL-8, G-CSF,
MCP-1, GM-CSF, RANTES, and TGF-
. The
neutralizing antibodies to human IL-8, G-CSF, MCP-1, RANTES, GM-CSF,
and TGF-
were purchased from Genzyme (Cambridge, MA). They were
added at the suggested concentrations to inhibit these cytokines to the
A549 cell supernatant fluids that were harvested after 72 h of
incubation with 10 µg/ml of bleomycin and incubated for 30 min at
37°C. Then these samples were used for chemotactic assay. To
evaluate the effect of IgG, nonimmune IgG was used as control.
Measurement of IL-8, G-CSF, MCP-1, GM-CSF, RANTES, and
TGF-
in the supernatant fluids. The
concentrations of IL-8, G-CSF, MCP-1, GM-CSF, RANTES, and TGF-
in
A549 cell supernatant fluids cultured for 72 h at a concentration of 10 µg/ml of bleomycin were measured with an enzyme-linked immunosorbent
assay (ELISA) according to the manufacturer's instructions. GM-CSF and
RANTES kits were purchased from Amersham, and the minimum
concentrations detected by these methods were 2.00 pg/ml for GM-CSF and
15.6 pg/ml for RANTES. IL-8, MCP-1, and TGF-
kits were purchased
from R&D Systems (Minneapolis, MN), and the minimum detectable
concentrations of IL-8, MCP-1, and TGF-
were 10.0 pg/ml, 31.3 pg/ml,
and 0.31 ng/ml, respectively. G-CSF kit was obtained from Chugai
Pharmaceutical (Tokyo, Japan). The minimum detectable concentration was
1.0 pg/ml.
Statistics. In experiments where
multiple experiments were done, differences between groups were tested
for significance with one-way analysis of variance, with Duncan's
multiple range test applied to data at specific time and dose points.
In experiments where a single measurement was made, the differences
between groups were tested for significance with Student's paired
t-test. In all cases, a
P value < 0.05 was considered
significant. Data are expressed as means ± SE.
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RESULTS |
Release of NCA and MCA from A549
cells. In response to bleomycin, A549 cells released
NCA and MCA in a dose-dependent manner (P < 0.05; Fig.
1). The lowest doses of
bleomycin to stimulate A549 cells were 0.1 µg/ml for neutrophils and
1 µg/ml for monocytes. Increasing concentrations up to 10 µg/ml of
bleomycin progressively increased the release of chemotactic
activity.

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Fig. 1.
Dose-dependent release of neutrophil
(A) and monocyte
(B) chemotactic activities in
response to bleomycin after 72 h of incubation. Values are means ± SE; n = 8 monolayers.
* P < 0.05 compared with
supernatant fluids without bleomycin.
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A549 cells released NCA and MCA in response to bleomycin in a
time-dependent manner (P < 0.05;
Fig. 2). After exposure to bleomycin, the
release of NCA increased significantly after 72 h and MCA increased
significantly after 48 h. Both NCA and MCA were cumulative even at 72 h. Bleomycin itself did not show any chemotactic activities for
neutrophils and monocytes (data not shown).

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Fig. 2.
Time-related release of neutrophil
(A) and monocyte
(B) chemotactic activities in
response to 10 µg/ml of bleomycin. , With bleomycin stimulation;
, without bleomycin stimulation. Values are means ± SE;
n = 8 monolayers.
* P < 0.05 compared with 12-h
supernatant fluids. ** P < 0.05 compared with supernatant fluids without bleomycin.
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The chemotactic responses to LTB4
at a concentration of 10
7 M
as a positive control were 1,020 ± 74 cells/10 HPF for neutrophils and 756 ± 34 cells/10 HPF for monocytes.
Checkerboard analysis revealed that the A549 cell supernatant fluids
stimulated by bleomycin induced neutrophil and monocyte migration with
increasing concentrations in the presence of a gradient across the
membrane (Table 1). Thus the
migration of neutrophils and monocytes was predominantly consistent
with chemotactic rather than chemokinetic activity.
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Table 1.
Checkerboard analysis of A549 cell culture supernatant fluid
harvested after 72 h in response to 10 µg/ml of
bleomycin
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Confirmation that the migrated cells were monocytes was provided by the
following lines of evidence: 1)
>90% of the migrated cells appeared to be monocytes morphologically
by light microscopy, 2) >90% of
the migrated cells were esterase positive, and
3) lymphocytes purified by allowing
the monocytes to attach to plastic and tested in the chemotaxis assay
yielded 0-20% of the chemotactic activity of the monocyte preparation.
Partial characterization of NCA and
MCA. NCA and MCA were heterogeneous in their character.
Both NCA and MCA were partially sensitive to heat, digested by trypsin,
and predominantly extractable into ethyl acetate (Fig.
3).

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Fig. 3.
Partial characterization of released neutrophil
(A) and monocyte
(B) chemotactic activities in
response to 10 µg/ml of bleomycin (BLEO) from A549 cells harvested
after 72 h of incubation. EA, ethyl acetate; RPMI, RPMI 1640 medium.
Values are means ± SE; n = 6 monolayers. * P < 0.05 compared with BLEO-exposed supernatant fluids.
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Molecular-sieve column chromatographic findings of the
released chemotactic activity. The released chemotactic
activities obtained from A549 cells that were incubated with 10 µg/ml
of bleomycin for 72 h were evaluated by molecular-sieve column
chromatography with Sephadex G-200. These experiments revealed that the
released NCA obtained from unstimulated cells was heterogeneous in size (Fig.
4A). At
least two peaks of activity were separated by column chromatography,
with the estimated molecular mass near that of cytochrome
c (molecular mass 12,300 Da) and an
additional peak that eluted near quinacrine (molecular mass 450 Da).
When stimulated with bleomycin, these two peaks became prominent.

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Fig. 4.
Molecular-sieve column chromatographic findings of released neutrophil
(A) and monocyte
(B) chemotactic activities in
response to 10 µg/ml of BLEO from A549 cells harvested after 72 h of
incubation. and solid lines, with BLEO stimulation; and dotted
lines, without BLEO stimulation. Nos. on
top and arrows, molecular mass and
position, respectively, of indicated markers.
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The MCA from unstimulated cells was also heterogeneous (Fig.
4B). At least three peaks of
activity were separated by column chromatography, with two peaks
between BSA and cytochrome c and an
additional peak near quinacrine. When stimulated with bleomycin, each
peak became prominent.
Effects of metabolic inhibitors on the release of
chemotactic activity. The supernatant fluids incubated
with 10 µg/ml of bleomycin in the presence of NDGA, DEC, and AA-861
showed a significant drop in the release of NCA and MCA. Cycloheximide
also inhibited the release of NCA and MCA in response to bleomycin
(P < 0.05; Fig.
5). NDGA, DEC, AA-861, and cycloheximide
did not inhibit the neutrophil migratory responses to activated serum,
fMLP, or A549 cell supernatant fluids harvested after 72 h of
incubation with 10 µg/ml of bleomycin (data not shown).

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Fig. 5.
Effects of nordihydroguaiaretic acid (NDGA), diethylcarbamazine (DEC),
AA-861, and cycloheximide (CYCLO) on release of neutrophil
(A) and monocyte
(B) chemotactic activities in
response to 10 µg/ml of BLEO from A549 cells harvested after 72 h of
incubation. Values are means ± SE;
n = 8 monolayers.
* P < 0.05 compared with
BLEO-exposed supernatant fluids.
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Effects of LTB4- and PAF-receptor
antagonists on chemotactic activity.
Both NCA and MCA were significantly inhibited by the addition of the
LTB4-receptor antagonist ONO-4057
(~50% for NCA and 40% for MCA; Fig. 6).
In contrast, the PAF-receptor antagonist TCV-309 did not inhibit NCA
and MCA. Each receptor antagonist at a concentration of
10
5 M inhibited neutrophil
migration in response to a
10
7 M concentration of
LTB4 and PAF but showed no
inhibitory effects on lipopolysaccharide-activated serum-induced
neutrophil and monocyte chemotaxis (data not shown).

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Fig. 6.
Effects of leukotriene (LT) B4
(ONO-4057)- and platelet-activating factor (PAF; TCV-309)-receptor
antagonists on released neutrophil
(A) and monocyte
(B) chemotactic activities in
response to 10 µg/ml of BLEO from A549 cells harvested after 72 h of
incubation. Values are means ± SE;
n = 8 monolayers.
* P < 0.05 compared with
BLEO-exposed supernatant fluids.
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Effects of bleomycin on the release of
LTB4 and PAF from A549 cells.
The measurement of LTB4 by RIA
revealed that A549 cells released
LTB4 in baseline culture
conditions. The addition of bleomycin at a concentration of 10 µg/ml
for 72 h induced a significant increase in
LTB4 release from A549 cells
(P < 0.05; Fig.
7). PAF was not detected in the supernatant
fluids.

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Fig. 7.
Release of LTB4 in response to 10 µg/ml of BLEO from A549 cells harvested after 72 h of incubation.
CONT, control. Values are means ± SE;
n = 6 monolayers.
* P < 0.05 compared with
supernatant fluids without BLEO.
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Effects of polyclonal antibodies to IL-8, G-CSF,
MCP-1, GM-CSF, RANTES, and TGF-
.
Because A549 cells had the potential to release chemotactic cytokines
and because chemotactic cytokines produced from A549 cells might be
responsible for the chemotactic activity, we used polyclonal blocking
antibodies to IL-8, G-CSF, MCP-1, GM-CSF, RANTES, and TGF-
. Among
these antibodies, anti-IL-8 and anti-G-CSF antibodies significantly
blocked NCA (Fig.
8A). Anti-MCP-1 antibody significantly blocked MCA (Fig.
8B). We evaluated the effects of
IL-8, G-CSF, MCP-1, and TGF-
antibodies on the column
chromatography-separated high-molecular-mass peaks of bleomycin-induced chemotactic activity. These antibodies inhibited the chemotactic activities at the corresponding molecular-mass peak (Table
2).

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Fig. 8.
Effects of anti-interleukin (IL)-8, anti-granulocyte colony-stimulating
factor (G-CSF), anti-transforming growth factor (TGF)- ,
anti-monocyte chemoattractant protein (MCP)-1, anti-released on
activation normal T cells expressed and secreted (RANTES), and
anti-granulocyte-macrophage colony-stimulating factor (GM-CSF)
polyclonal antibodies on released neutrophil
(A) and monocyte
(B) chemotactic activity in response
to 10 µg/ml of BLEO from A549 cells incubated for 72 h. Values are
means ± SE; n = 6 monolayers.
* P < 0.05 compared with
BLEO-exposed supernatant fluids.
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Table 2.
Effects of specific antibodies and LTB4-receptor
antagonist on column chromatography-separated neutrophil and
monocyte chemotactic peaks
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Anti-IL-8 and anti-G-CSF antibodies inhibited the neutrophil migratory
response to recombinant human IL-8 and G-CSF completely but did not
inhibit the neutrophil migratory response to activated serum and fMLP.
Anti-MCP-1 antibody also inhibited the monocyte migratory response to
human recombinant MCP-1 but not to fMLP and activated serum. Nonimmune
IgG did not inhibit the neutrophil and monocyte migratory responses to
supernatant fluid harvested after 72 h of incubation with 10 µg/ml of bleomycin.
Effects of bleomycin on the release of IL-8, G-CSF,
MCP-1, GM-CSF, RANTES, and TGF-
from A549
cells. A549 cells released detectable amounts of IL-8,
G-CSF, MCP-1, and TGF-
constitutively (Fig.
9). A549 cells stimulated by bleomycin at a
concentration of 10 µg/ml released IL-8, G-CSF, and MCP-1
significantly compared with the unstimulated supernatants. Bleomycin
did not stimulate TGF-
significantly (Fig.
9D). RANTES and GM-CSF were not
detected in A549 cell supernatant fluids in both the unstimulated and
stimulated supernatant states.

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Fig. 9.
Release of IL-8 (A), G-CSF
(B), MCP-1
(C), and TGF-
(D) in response to 10 µg/ml of
BLEO from A549 cells incubated for 72 h. Values are means ± SE;
n = 6 monolayers.
* P < 0.05 compared with
supernatant fluids without BLEO stimulation
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DISCUSSION |
The present study demonstrated that bleomycin stimulated A549 cells to
release NCA and MCA in a dose- and time-dependent manner. Partial
characterization and molecular-sieve column chromatography revealed the
heterogeneity of NCA and MCA. Anti-IL-8 and anti-G-CSF antibodies and
the LTB4-receptor antagonist
inhibited NCA. Anti-MCP-1 antibody and the
LTB4-receptor antagonist inhibited
MCA. Although A549 cells released IL-8, G-CSF, MCP-1, and
LTB4 constitutively, bleomycin
significantly stimulated the release of IL-8, G-CSF, MCP-1, and
LTB4. These data suggest that an
interaction between alveolar epithelial cells and bleomycin may
modulate inflammatory cell recruitment to the alveolar space.
Because A549 cells are a cell line derived from an adenocarcinoma of
the lung, release and gene regulation may be particularly upregulated.
However, two reports (19, 33) described that the human primary type II
alveolar epithelial cells and human primary bronchial epithelial cells
released IL-8 in response to smoke extract. In these reports, the
primary type II alveolar epithelial cells and bronchial epithelial
cells responded to smoke extract in a manner similar to A549 cells
(18). Furthermore, rat type II alveolar epithelial cells in primary
culture responded in a manner similar to A549 cells to IL-1 and TNF,
releasing MCP-1 and GM-CSF (3, 20). The releasing potential of MCP-1
and GM-CSF from rat primary alveolar type II cells, the major MCA in
the present study, was very similar to the A549 cell response to IL-1
and TNF (unpublished observations). Then we speculated that A549 cells responded similarly to IL-1 and TNF as did rat primary
type II alveolar epithelial cells. Thus the human and rat primary
alveolar type II epithelial cells as well as the A549 cells were highly
upregulated for the production of cytokines.
Inflammatory cell recruitment is a significant event in interstitial
lung diseases that leads to pulmonary fibrosis. Bleomycin-induced lung
injury involves a pulmonary inflammatory response characterized by
increases in mononuclear cells and granulocytes. The mechanisms by
which inflammatory cells recruit to the lung remain to be elucidated. The generation of chemotactic factors that direct neutrophils and
monocytes to the interstitium and alveolar space seems essential. The
experimental studies have focused on immune competent cells and
fibroblasts as key sources associated with bleomycin-induced lung
inflammation. However, the role of alveolar type II epithelial cells
has so far received little attention.
Type II alveolar epithelial cells exist in the lung as a highly
differentiated phenotype with numerous specialized functions, including
production of surfactants, differentiation to type I alveolar
epithelium, and release of cytokines and growth factors. In the present
study, we demonstrated that type II alveolar epithelial cells released
NCA and MCA in response to bleomycin. The release of NCA and MCA in the
supernatant fluids was significantly increased compared with that in
control fluids. The released NCA and MCA were almost similar to or more
than the released activity from alveolar macrophages at
106 cells/ml in response to
Escherichia coli lipopolysaccharide
(data not shown). Thus type II epithelial cells are one of the
important cell sources for the release of NCA and MCA.
The released NCA and MCA in response to bleomycin stimulation appear to
be predominantly lipid extractable and of low molecular mass. The
release was inhibited by lipoxygenase inhibitors. The LTB4-receptor antagonist inhibited
the chemotactic activity. The concentration of
LTB4 in the supernatant fluids
increased and reached a concentration for neutrophil and monocyte
chemotaxis. Thus LTB4 is the
predominant chemotactic activity in culture supernatant fluids of A549
cells stimulated by bleomycin.
Partial characterization of NCA and MCA showed that the released
activities were, in part, heat and trypsin sensitive, and the release
of NCA and MCA was inhibited by cycloheximide. These data suggest that
the chemotactic activities were partly composed of protein.
Molecular-sieve column chromatography of NCA and MCA revealed that
there were high-molecular-mass peaks of chemotactic activities in
response to bleomycin. Because the released NCA and MCA were inhibited
by specific antibodies to IL-8, G-CSF, and MCP-1, these cytokines were
responsible for peptide chemotactic activity. The production of these
cytokines by A549 cells has been reported in response to TNF-
, IL-1,
and asbestos (25, 28). In the present study, IL-8, G-CSF, and MCP-1
were released from A549 cells constitutively. Bleomycin further
augmented release of these cytokines from A549 cells significantly.
These findings may suggest that type II alveolar epithelial cells
exposed to bleomycin may have the potential to release IL-8, G-CSF, and
MCP-1 as NCA and MCA.
Although NDGA at a concentration of 100 µM inhibited the release of
LTB4 almost completely, NDGA also
inhibited the release of IL-8 significantly. The addition of DEC and
AA-861 did not inhibit the release of IL-8. The addition of
cycloheximide at 10 µg/ml completely inhibited the release of IL-8
and slightly but significantly inhibited the release of
LTB4 from A549 cells in response
to bleomycin after 72 h of incubation. In these points, NDGA and
cycloheximide were not specific appropriate inhibitors. However, the
effects of specific receptor antagonists and antibodies and the direct
measurement of LTB4 and each
cytokine may support the release of
LTB4 and cytokines from A549 cells
in response to bleomycin.
TGF-
is a multifunctional mediator capable of regulating cell
proliferation and differentiation as well as synthesis of many components of the extracellular matrix (27). It has been associated with fibrotic processes in many organs including the lung. TGF-
can
induce monocyte chemotaxis at concentrations from 0.1 to 10 pg/ml (24).
In the present study, A549 cells released TGF-
constitutively.
Bleomycin did not augment the release of TGF-
significantly.
Although the concentration of TGF-
in the supernatant fluids was
more than the chemotactic concentration for monocytes, the proteolytic
processing of TGF-
is needed for the activation of TGF-
. In the
present experiment, the activation of TGF-
released from A549 cells
is uncertain. The activation may not be enough for monocyte chemotaxis.
In conclusion, bleomycin stimulated A549 cells to release NCA and MCA.
The released activities were chemotactic by checkerboard analysis and
heterogeneous in their character. NCA and MCA included LTB4, IL-8, G-CSF, and MCP-1.
These results suggest that bleomycin may play a role in inflammatory
cell recruitment by stimulating lung epithelial cells to release NCA
and MCA.
 |
FOOTNOTES |
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: S. Koyama,
The First Dept. of Internal Medicine, Shinshu Univ. School of Medicine,
3-1-1 Asahi, Matsumoto, 390 Japan.
Received 21 April 1998; accepted in final form 22 February 1999.
 |
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