Bleomycin stimulates lung fibroblast and epithelial cell lines to

Copyright #ERS Journals Ltd 2000
European Respiratory Journal
ISSN 0903-1936
Eur Respir J 2000; 16: 951±958
Printed in UK ± all rights reserved
Bleomycin stimulates lung fibroblast and epithelial cell lines to
release eosinophil chemotactic activity
E. Sato*, S. Koyama**, R.A. Robbins*
Bleomycin stimulates lung fibroblast and epithelial cell lines to release eosinophil
chemotactic activity. E. Sato, S. Koyama, R.A. Robbins. #ERS Journals Ltd 2000.
ABSTRACT: The presence of eosinophils in the lungs of patients with pulmonary
fibrosis correlates with poor prognosis or resistance to therapy. Furthermore,
eosinophils localize to areas undergoing active fibrosis. It was hypothesized that a
human lung fibroblast (HFL-1) and a human lung epithelial cell line (BEAS-2B) might
release eosinophil chemotactic activity (ECA) in response to bleomycin, a chemotherapeutic agent associated with pulmonary fibrosis.
HFL-1 and BEAS-2B cells were cultured in the presence of bleomycin and their
supernatant fluids evaluated for ECA by means of a Boyden chamber method.
HFL-1 and BEAS-2B cells released ECA in a dose- and time-dependent manner in
response to bleomycin, and partial characterization revealed that the ECA was
heterogeneous. ECA release from HFL-1 and BEAS-2B cells was significantly reduced
by a leukotriene B4 (LTB4) receptor antagonist and an antibody directed against
granulocyte-macrophage colony-stimulating factor. HFL-1 cells released LTB4,
eotaxin, and GM-CSF constitutively, and BEAS-2B cells released LTB4, eotaxin,
regulated on activation, normal T-cell expressed and presumably secreted, and GMCSF constitutively. In both cases, the release of GM-CSF was significantly increased in
response to bleomycin.
These data suggest that lung fibroblasts and epithelial cells may modulate
eosinophil recruitment into the lung in bleomycin-induced pulmonary fibrosis.
Eur Respir J 2000; 16: 951±958.
Several studies have suggested that eosinophils may
play a role in pulmonary fibrosis. An enhanced number
of eosinophils is often found in the interstitium and
alveolar spaces in lung biopsy specimens obtained from
patients with pulmonary fibrosis, including those induced
by bleomycin [1±3]. The influx of eosinophils into the
pulmonary compartment has also been demonstrated in
bronchoalveolar lavage fluid (BALF) from patients with
idiopathic pulmonary fibrosis, and a higher number of
eosinophils in BALF has been related to a poor response
to therapy and shorter survival [4±6]. In animal models of
bleomycin-induced pulmonary fibrosis, infiltration by
eosinophils precedes and parallels the development of the
fibrotic lesion [7].
The concept that eosinophils can contribute to lung
injury is not new [8]. Mechanisms of eosinophil influx
into the lungs have increasingly emphasized the role of
bronchial epithelial cells and lung fibroblasts in releasing
a variety of eosinophil chemoattractants, including eotaxin, granulocyte-macrophage colony-stimulating factor
(GM-CSF), interleukin (IL)-5, regulated on activation,
normal T-cell expressed and presumably secreted
(RANTES) and leukotriene B4 (LTB4) [9±12]. In vitro
studies have demonstrated that migration of eosinophils is
induced by these chemoattractants [13, 14], and in vivo
studies show the importance of cytokine mobilization in
*Research Service, Southern Arizona
Veterans Health Care System, and Dept
of Medicine, University of Arizona,
Tucson, AZ, USA. **First Dept of
Internal Medicine, Shinshu University
School of Medicine, Matsumoto, Japan.
Correspondence: R.A. Robbins, Research
Health Care Group, Tucson Veterans
Administration Medical Center, 3601 S.
6th Avenue, Tucson, AZ 85723, USA.
Fax: 1 5206291801
Keywords: Chemotaxis
eosinophil
eotaxin
granulocyte-macrophage colonystimulating factor
leukotriene B4
regulated on activation, normal T-cell
expressed and presumably secreted
Received: October 20 1999
Accepted after revision July 20 2000
promoting airway eosinophil recruitment in pulmonary
fibrosis [15]. Consistent with the importance of cytokines,
the levels of IL-5, GM-CSF and RANTES in BALF and
messenger ribonucleic acid expression in lung were
increased in idiopathic pulmonary fibrosis patients and
bleomycin-induced pulmonary fibrosis animal models
[16±19]. Antibodies directed against IL-5 or tumour
necrosis factor-a caused a significant reduction in lung
eosinophilia and fibrosis in bleomycin-induced pulmonary
fibrosis [20, 21]. Furthermore, transfer of a GM-CSF gene
with high expression into rat lung induced eosinophilia
and fibrosis in rat models [22].
Bleomycin is one of the antitumour antibiotics produced
by Streptomyces verticullis [23]. Bleomycin-induced fibrosis limits the usefulness of the drug, and bleomycininduced fibrosis resembling the human disease process
has been produced in a variety of animal models [24, 25].
On the basis of these observations, definition of the
mediators and cytokines that participate in the recruitment
of eosinophils into the lungs in bleomycin-induced
pulmonary fibrosis was attempted. The results demonstrate that a human lung fibroblast (HFL-1) and a human
lung epithelial cell lines (BEAS-2B) release eosinophil
chemotactic activity (ECA), but, of the chemotactic
factors assayed, only GM-CSF release was significantly
increased by bleomycin.
952
E. SATO ET AL.
Materials and methods
Culture of human foetal lung fibroblasts and BEAS-2B
cells
The human foetal lung fibroblast cell line HFL-1
(diploid, passage 14), was purchased from the American
Type Culture Collection (ATCC, Rockville, MD, USA).
This cell line was initiated from the lung tissue of a 16±18week-old human foetus [26]. The morphology of HFL-1
cells is fibroblast-like and they retain features of normal
lung fibroblasts including collagen and fibronectin production [27]. BEAS-2B, a human bronchial epithelial cell
line transformed by the hybrid adenovirus simian vacuolating virus 40, was also purchased from the ATCC [28].
The HFL-1 and BEAS-2B cells were suspended at
1.06106 cells.mL-1 in Ham's F-12 or Dulbecco modified
Eagle medium (DMEM; Gibco, Grand Island, NY, USA)
supplemented with penicillin (50 mg.mL-1; Gibco), streptomycin (50 mg.mL-1; Gibco) and 10% heat-inactivated
foetal bovine serum. Cell suspensions (3 mL) were added
to 30-mm-diameter tissue culture dishes (Corning, Corning, NY, USA) and cultured under a 5% CO2 atmosphere at
378C. After 2±3 days in culture, the cells reached confluence and the culture medium was replaced with 2 mL
medium supplemented as above and incubated one further
day.
In order to confirm the results obtained with the HFL-1
and BEAS-2B cell lines, the cell lines CCL-202 and A549
(both purchased from the ATCC), which are a human lung
fibroblast cell line and a human lung epithelial cell line
respectively, were also studied. These cell lines were
grown in DMEM under conditions similar to the HFL-1
cells until confluent.
Exposure of lung HFL-1 and BEAS-2B cells to bleomycin
Culture medium was removed from cells by washing
twice with serum-free medium, and the cells were incubated in the presence and absence of bleomycin. In order
to determine the dose- and time-dependent release of
ECA, the cultures were incubated at various concentrations
of bleomycin (0, 0.01, 0.1, 1.0 and 10 mg.mL-1, 1.5 U=1
mg; Sigma, St Louis, MO, USA) in a humidified 5% CO2
atmosphere for 12, 24, 48 and 72 h at 378C. Cell injury
was evaluated by microscopy (cell shape and detachment
from tissue culture dish) and trypan blue exclusion. The
supernatant fluids were then harvested and stored at -808C
until assay. At least six separate HFL-1 and BEAS-2B cell
supernatant fluids were harvested for each experimental
condition.
Measurement of eosinophil chemotactic activity
Eosinophils were isolated using a modified method of
HANSEL et al. [29] with magnetic cell separation. Briefly,
venous blood anticoagulated with 130 mM trisodium
citrate was obtained from normal human volunteers and
diluted with phosphate-buffered saline (0.9% saline,
pH7.1) in a 1:1 ratio. Diluted blood was overlaid on an
isotonic Percoll solution (density 1.082 g.mL-1; Sigma)
which was then centrifuged for 30 min at 1,0006g at 48C
in a Beckman TJ-6 centrifuge (Beckman Coulter, Fullerton, CA, USA). The supernatant and mononuclear cells
at the interface were carefully removed, and red blood
cells in the pellet were lysed with two cycles of hypotonic
lysis (0.1% KHCO3; 0.83% NH4Cl). Isolated granulocytes were washed twice with piperazine-N, N'-bis (2ethanesulphonic acid (PIPES) buffer (25 mM PIPES, 50
mM NaCl, 5 mM KCl, 25 mM NaOH, 5.4 mM glucose,
pH7.4) containing 1% defined calf serum (DCS; Hyclone
Laboratories, Logan, UT, USA), and an approximately
equal volume of anti-CD16 conjugated with magnetic
particles (Miltenyi Biotec, Bergisch Gladbach, Germany)
was added to the cell pellet. After a 60-min incubation
on ice, 5 mL PIPES buffer containing 1% DCS were
added to the cell/antibody mixture. The resuspended cells
were loaded on to the separation column positioned in
the magnetic cell separation system (miltenyi) with a
strong magnetic field. The cells were eluted three times
with 5 mL PIPES buffer containing 1% DCS, according
to the manufacturer's instructions. The purity of the
eosinophils counted using Randolph's stain was >94%;
the viability was >98%. The eosinophils were resuspended in Gey's solution at 2.06106 cells.mL-1 and
used for the chemotaxis assay.
The chemotaxis assay was performed in a 48-well
microchemotaxis chamber (NeuroProbe, Cabin John, MD,
USA) using a modification of a previously described
technique [30]. The bottom wells of the chamber were
filled with 25 mL fluid containing the chemotactic
stimulus or medium in duplicate. A 10-mm-thick polyvinylpyrrolidone-free polycarbonate filter (Nucleopore,
Pleasanton, CA, USA), with a pore size of 5 mm, was
placed over the bottom wells. The silicone gasket and
upper pieces of the chamber were applied, and 50 mL cell
suspension was placed into the upper wells above the
filter. The chambers were incubated in humidified air in
5% CO2 for 90 min at 378C. After incubation, the
chamber was disassembled and nonmigrated cells were
wiped from the filter. The filter were fixed and stained
using a modified Wright's stain and mounted on a glass
slide. Cells that had completely migrated through the
filter were counted using light microscopy in random
high-power fields (HPFs, 6400).
Partial characterization of eosinophil chemotactic
activity
Partial characterization of the ECA released from HFL-1
or BEAS-2B cells was performed using supernatant fluids
harvested after 72 h at 10 mg.mL-1 bleomycin; the protein
or lipid factor in the supernatant fluid was measured.
Sensitivity to proteases was tested by incubating the
supernatant fluids with trypsin (100 mg.mL-1, Sigma) for
30 min at 378C; this was followed by the addition of a 1.5
M excess of soybean trypsin inhibitor (Sigma) to terminate
the proteolytic activity, and then chemotactic activity was
evaluated [31]. The lipid solubility was evaluated by
mixing the supernatant fluids twice with ethylacetate,
decanting the lipid phase after each extraction, evaporating the ethylacetate to dryness and resuspending the
extracted material in the medium used for cell culture
before the chemotaxis assay. Both extracted materials and
the lipid-insoluble phase were evaluated for chemotactic
activity. Heat sensitivity was determined by maintaining a
supernatant fluid at 988C for 15 min.
953
BLEOMYCIN-INDUCED EOSINOPHIL CHEMOTACTIC ACTIVITY RELEASE
LTB4 was measured in the culture supernatant fluids
because the released ECA was only partially lipid
extractable. The concentrations of LTB4 in HFL-1 or
BEAS-2B cell supernatant fluids cultured for 72 h at 10
mg.mL-1 bleomycin were measured using a commercially
available enzyme-linked immunosorbant assay (ELISA)
(R&D Systems, Minneapolis, MN, USA). The detection
limit of this method is 30 pg.mL-1.
Measurement of eotaxin, interleukin-5, regulated on
activation, normal T-cell expressed and presumably
secreted, and granulocyte-macrophage, colony-stimulating factor in the supernatant fluids
Several well-characterized protein chemotactic factors
were measured in the culture supernatant fluids because the
released ECA was partially heat- and protease-sensitive.
The concentrations of eotaxin, IL-5, RANTES and GMCSF in HFL-1 or BEAS-2B cell supernatant fluids cultured
for 72 h at 10 mg.mL-1 bleomycin were measured using a
commercially available ELISA (R&D Systems). The
detection limit of these methods was ~15 pg.mL-1.
Effects of leukotriene B4 and platelet-activating factor
receptor antagonists on eosinophil chemotactic activity
LTB4 and platelet-activating factor (PAF) receptor antagonists were used to evaluate the involvement of LTB4
and PAF in the supernatant fluids of cells stimulated with
bleomycin, because they were extractable into ethylacetate
and LTB4 levels were found to be elevated. An LTB4
receptor antagonist (ONO 4057; ONO Pharmaceutical,
Tokyo, Japan) and PAF receptor antagonist (TCV309;
Takeda Pharmaceutical, Tokyo, Japan), each at a concentration of 10-5 M, were used to evaluate the involvement of
LTB4 and PAF as ECA in the crude supernatant fluids [32,
33].
Analysis
In experiments in which multiple measurements were
made, differences between groups were tested for significance using the Kruskal-Wallis nonparametric test. In
experiments in which a single measurement was made, the
differences between groups were tested for significance
using the Wilcoxon ranked sum test. In all cases, a p-value
of <0.05 was considered significant. Data in figures and
tables are presented as mean‹SEM.
Results
Release of eosinophil chemotactic activity from lung
fibroblast and epithelial cell lines
HFL-1 and BEAS-2B cells released ECA in a dosedependent manner in response to bleomycin (fig. 1). The
lowest doses of bleomycin to stimulate HFL-1 and
BEAS-2B cells were 0.1 and 1 mg.mL-1, respectively.
Increasing concentrations of bleomycin up to 10 mg.mL-1
progressively increased the release of ECA. Bleomycin
did not cause HFL-1 or BEAS-2B cell injury (no deformity of cell shape, no detachment from tissue culture
dish and >95% cell viability by trypan blue exclusion)
after 72 h incubation with 10 mg.mL-1 bleomycin.
However, bleomycin at 100 mg.mL-1 caused substantial
cytotoxicity after 24 h of incubation.
a) 35
30
ECA cells·HPF-1
Measurement of leukotriene B4 in the supernatant
fluids
*
*
*
25
20
15
10
5
0
Neutralizing antibodies to eotaxin, IL-5, RANTES and
GM-CSF (5, 10, 50 and 10 mg.mL-1, respectively, all R&D
Systems) were added to the supernatant fluids which were
harvested after 72 h at 10 mg.mL-1 bleomycin to inhibit
these cytokines and incubated for 30 min at 378C. Then
these samples were used for chemotactic assay. As a
negative control, nonimmune immunoglobulin G (IgG)
was used. These antibodies reduced the ECA of recombinant cytokines (0.02 mg.mL-1 eotaxin, 0.1 ng.mL-1
IL-5, 0.1 mg.mL-1 RANTES, and 0.05 ng.mL-1 GM-CSF
(all R&D Systems)) to baseline levels (data not shown).
They did not influence the chemotactic response to
endotoxin-activated serum and LTB4 (data not shown).
b) 25
*
*
20
ECA cells·HPF-1
The effects of eotaxin, interleukin-5, regulated on activation, normal T-cell expressed and presumably secreted, and granulocyte-macrophage colony-stimulating
factor polyclonal antibodies to eosinophil chemotactic
activity
15
10
5
0
0
0.1
0.01
Bleomycin µg·mL-1
1
10
Fig. 1. ± Dose-dependent release of eosinophil chemotactic activity
(ECA) from: a) HFL-1; and b) BEAS-2B cells after 72 h incubation with
bleomycin (n=8). Data are presented as mean‹SEM. HPF: high-power
field. *: p<0.05 versus supernatant fluids without bleomycin.
954
E. SATO ET AL.
a) 35
*
ECA cells·HPF-1
30
25
20
*
Partial characterization of eosinophil chemotactic
activity
15
10
5
0
b) 30
*
ECA cells·HPF-1
25
The ECA from HFL-1 or BEAS-2B cells was
heterogeneous in character. It was partially sensitive to
heat, extracted by ethylacetate and digested by trypsin
(p<0.05) (fig. 3). Trypsin and soybean trypsin inhibitor
had no effect on the ECA induced by LTB4 (p>0.05).
Release of leukotriene B4 from HFL-1 and BEAS-2B
cells
20
15
10
5
0
in response to 10 mg.mL-1 bleomycin than when cultured
in medium alone (CLL-202: 6.2‹1.1 versus 26.7‹1.5
cells.HPF-1; A549: 14‹3.2 versus 46‹4.5 cells.HPF-1, n=4,
p<0.05 for both comparisons).
0
10
20
50
30
40
Incubation time h
60
70
Fig. 2. ± Time course of eosinophil chemotactic activity (ECA) release
from: a) HFL-1; and b) BEAS-2B cells in response to 10 mg.mL-1
bleomycin (s ; n=8). * : cultures without bleomycin (n=8). Data are
presented as mean‹SEM. HPF: high-power field. *: p<0.05 versus
supernatant fluids without bleomycin.
Although HFL-1 and BEAS-2B cells released ECA
constitutively, release was further increased in response to
bleomycin in a time-dependent manner. The release of
ECA was significant after a 48-h exposure to bleomycin for
HFL-1 cells and a 72-h exposure for BEAS-2B cells (fig.
2). Bleomycin itself did not show any ECA (data not
shown).
These results were confirmed in CLL-202 and A549
cells. Both had released significantly more ECA after 48 h
a) 35
Measurement of LTB4 level in supernatant fluids by
ELISA revealed that HFL-1 and BEAS-2B cells released
significant amounts of LTB4 under baseline culture conditions. However, addition of bleomycin at 10 mg.mL-1
for 72 h did not increase LTB4 release significantly from
HFL-1 cells (725.1‹64.6 pg.mL-1 in controls versus
821.2‹47.6 pg.mL-1 with bleomycin) or BEAS-2B cells
(938.6‹116.2 pg.mL-1 in controls versus 822.7‹74.2
pg.mL-1 with bleomycin).
Release of eotaxin, interleukin-5, regulated on activation, normal T-cell expressed and presumably secreted,
and granulocyte-macrophage colony-stimulating factor
from HFL-1 and BEAS-2B cells by bleomycin
Measurement of chemotactic cytokines by ELISA
revealed that incubation with 10 mg.mL-1 bleomycin for
72 h stimulated the release of GM-CSF from HFL-1 and
BEAS-2B cells (p<0.05) (table 1). HFL-1 cells released
eotaxin and BEAS-2B cells released eotaxin and
RANTES under baseline culture conditions; however,
addition of bleomycin at 10 mg.mL-1 for 72 h did not
increase the release of these factors.
b)
ECA cells·HPF-1
30
25
*
20
*
15
*
*
*
EA
TD
*
*
*
LI
EA
10
5
0
CSF
H
LI
F12
CSF
H
TD DMEM
Fig. 3. ± Partial characterization of eosinophil chemotactic activity (ECA) obtained from: a) HFL-1; and b) BEAS-2B cells after 72 h incubation with 10
mg.mL-1 bleomycin or medium alone (n=8). HPF: high-power field; CSF: crude supernatant fluid; H: heat-treated supernatant; LI: lipid-insoluble phase;
EA: ethylacetate extract; TD: trypsin digest; F12: Ham's F12 medium; DMEM: Dulbecco modified Eagle medium. Data are presented as mean‹SEM. *:
p<0.05 versus CSFs.
955
BLEOMYCIN-INDUCED EOSINOPHIL CHEMOTACTIC ACTIVITY RELEASE
Table 1. ± Release of cytokines from HFL-1 and BEAS-2B cells in response to bleomycin
HFL-1
Eotaxin pg.mL-1
RANTES pg.mL-1
IL-5 pg.mL-1
GM-CSF pg.mL-1
LTB4 pg.mL-1
BEAS-2B
Control
Bleomycin
Control
Bleomycin
691.8‹38.4
ND
ND
17.9‹0.3
725.1‹64.6
546.87‹38.4
ND
ND
74.5‹11.2*
821.2‹47.6
25.0‹1.5
62.5‹12.4
ND
19.17‹0.4
938.6‹116.2
20.22‹1.7
61.4‹13.3
ND
54.6‹10.6*
822.7‹74.2
Data are presented as mean‹SEM. RANTES: regulated on activation, normal T-cell expressed and presumably secreted; IL-5:
interleukin-5; GM-CSF: granulocyte-macrophage colony-stimulating factor; LTB4: leukotriene B4; ND: not determined. *: p<0.05
versus control.
Inhibition of eosinophil chemotactic activity release by
leukotriene B4 receptor antagonists
ECA release from HFL-1 and BEAS-2B cells was
significantly inhibited by addition of the LTB4 receptor
antagonist, ONO 4057 (p<0.05) (fig. 4). The PAF receptor
antagonist, TCV309, did not block ECA release. LTB4 or
PAF receptor antagonist at a concentration of 10-5 M
inhibited ECA release in response to 10-7 M LTB4 or PAF
but showed no inhibitory effects on endotoxin-activated
serum-induced eosinophil chemotaxis (data not shown).
Inhibition of eosinophil chemotactic activity by polyclonal antibodies to eotaxin, interleukin-5, regulated on
activation, normal T-cell expressed and presumably secreted, and granulocyte-macrophage colony-stimulating
factor
Polyclonal blocking antibodies were used to determine
the contribution of identified protein eosinophil chemotactic factors. Among these antibodies, GM-CSF antibodies inhibited ECA release from HFL-1 and BEAS-2B
cells (p<0.05) (fig. 5). IL-5 and RANTES antibodies did
not show any effect on ECA release. Nonimmune IgG did
not have any effect on bleomycin-conditioned medium
(data not shown).
Discussion
The present study demonstrates that bleomycin stimulates HFL-1 and BEAS-2B cells to release ECA in a doseand time-dependent manner. Partial characterization
a) 40
revealed the heterogeneity of the ECA. LTB4 and eotaxin
were released from HFL-1 cells constitutively. LTB4,
eotaxin and RANTES were released from BEAS-2B cells
constitutively. However, release of these factors did not
increase in response to bleomycin. In contrast, release of
GM-CSF from HFL-1 and BEAS-2B cells was increased
by bleomycin stimulation. These data suggest that an
interaction between fibroblasts or epithelial cells and
bleomycin may modulate eosinophil recruitment to the
interstitium after bleomycin exposure through the generation of chemotactic cytokines.
Eosinophils are known to be a key source of cytokines
with inflammatory and fibrosis-promoting activities [34].
Consistent with their potential role in the promotion of
fibrosis, eosinophils have been found to localize in areas
undergoing active fibrosis, expressing such fibrosispromoting cytokines as transforming growth factor-b1
[7]. Furthermore, the presence of eosinophils in the lungs
of patients with idiopathic pulmonary fibrosis correlates
with a worse prognosis or resistance to therapy [4±6].
Thus there is compelling evidence to suggest that eosinophils may play an important role in pulmonary fibrosis.
Although several types of lung cell can produce
chemotactic cytokines, production by airway epithelial
cells and fibroblasts may be particularly relevant to
pulmonary fibrosis. It is becoming increasingly clear that
these cells participate in inflammatory processes by producing a variety of mediators and cytokines, including
prostaglandins, PAF, IL-3, IL-5, IL-8, macrophage inflammatory protein 1a, monocyte chemoattractant protein
(MCP)-3, MCP-4, RANTES, eotaxin, and GM-CSF
b)
ECA cells·HPF-1
35
30
25
*
20
*
15
10
5
0
BLEO
BLEO + BLEO +
ONO 4057 TCV309
F12
BLEO
BLEO + BLEO +
ONO 4057 TCV309
DMEM
Fig. 4. ± Inhibition of eosinophil chemotactic activity (ECA) release from: a) HFL-1; and b) BEAS-2B cells by the leukotriene B4 receptor antagonist,
ONO 4057, and the platelet-activating factor receptor antagonist, TCV309 (n=8). As a negative control, cells were incubated with medium alone. Data
are presented as mean‹SEM. *: p<0.05 versus crude supernatant fluids.
956
E. SATO ET AL.
a) 40
b)
ECA cells·HPF-1
35
30
25
20
*
*
15
10
5
0
USF GM-CSF IL-5
USF GM-CSF IL-5
RANTES Eotaxin F12
RANTES Eotaxin DMEM
Fig. 5. ± Effects of blocking antibodies on eosinophil chemotactic activity (ECA) from: a) HFL-1; and b) BEAS-2B cells (n=8). All cell cultures were
incubated in the presence of 10 mg.mL-1 bleomycin on medium alone prior to the addition of antibodies directed against the indicated cytokines. Data are
presented as mean‹SEM. GM-CSF: granulocyte-macrophage colony-stimulating factor; IL-5: interleukin-5; RANTES: regulated on activation, normal Tcell, expressed and presumably secreted. *: p<0.05 versus untreated supernatant fluids (USFs).
[9±12, 35±38]. On the basis of these observations,
definition of the mediators involved in bleomycin-induced
pulmonary fibrosis was attempted.
The cell lines HFL-1 and BEAS-2B were used in the
present studies because of the difficulty in obtaining
enough samples for experiments from humans. The bleomycin effects were also investigated with another human
lung fibroblast cell line (CCL-202, a fibroblast-like cell
line derived from normal lung tissue) and A549 cells (type
II epithelial cell line derived from an individual with
alveolar carcinoma) to confirm the results. Bleomycin
stimulated CCL-202 and A549 cells to release ECA.
Bleomycin is one of the antitumour antibiotics produced
by Streptomyces verticullis, discovered in 1966 by
UMEZAWA et al. [23]. It has been used in the treatment
of a variety of neoplasms; however, bleomycin-induced
pneumonitis or pulmonary fibrosis is sometimes a fatal
complication [39]. FUJITA et al. [40] reported that the
concentrations of bleomycin in blood and cancer tissue
obtained from humans administrated 15 mg intravenously
were 0.8 and 0.4 mg.mL-1. UMEZAWA et al. [41] reported
higher concentrations of bleomycin in the lungs (18 mg.
g-1) compared with other organs, which may be related to
the lung side-effects of this drug. It is possible that
bleomycin concentrations reach these experimental levels
around the epithelial cells or fibroblasts in the lung.
The results of partial characterization suggest that the
ECA released by HFL-1 and BEAS-2B cells is partly
composed of lipid. Consistent with these observations,
LTB4 concentrations detected in the culture supernatant
fluids were in the chemotactic range for eosinophils [42]
and ECA was inhibited by a LTB4 receptor antagonist.
Although the release of LTB4 from HFL-1 or BEAS-2B
cells in response to bleomycin was not significantly
increased compared with controls, LTB4 is one of the
chemoattractants for eosinophils released from fibroblasts
and epithelial cells constitutively.
The results of partial characterization also indicate that
the released activities are partially protein in nature.
Although eotaxin, RANTES and GM-CSF were detected
in the culture supernatant fluids and blocking antibodies
decreased ECA, GM-CSF was the only cytokine released
in response to bleomycin. These data suggest that GMCSF is an important constituent of the ECA released by
HFL-1 and BEAS-2B cells in response to bleomycin.
GM-CSF has been shown to activate eosinophil
functions including differentiation, chemotaxis, chemotaxis
priming, survival, transendothelial migration, expression
of CD11b, CD4 and histocompatibility leukocyte antigen
DR, and mediator release in vitro [43, 44]. GM-CSF was
initially purified from mouse lung conditioned medium,
thus indicating that the lung is an important producer of
GM-CSF [45]. This cytokine can be produced by a wide
spectrum of cell types of the lung, including fibroblasts
and epithelial cells, and significantly increased levels are
detected in BALF or lung tissues obtained from patients
with idiopathic pulmonary fibrosis [18, 19].
Recently XING et al. [22] reported that overexpression
of GM-CSF by means of a gene transfer technique
induces eosinophilia and pulmonary fibrotic reactions.
GM-CSF induces accumulation of both eosinophils and
macrophages in the early stages and irreversible fibrotic
reaction later on. In addition, GM-CSF has been found to
induce fibrotic responses with accumulation of myofibroblasts after chronic subcutaneous administration of
GM-CSF [46]. These findings support the notion of
GM-CSF as a fibrogenic cytokine.
In conclusion, bleomycin stimulated both HFL-1 and
BEAS-2B cells to release eosinophil chemotactic activity.
The released eosinophil chemotactic activity was heterogeneous, but granulocyte-macrophage colony-stimulating
factor was the only eosinophil chemotactic factor whose
concentration was significantly increased by bleomycin
stimulation. These results suggest that lung fibroblasts and/
or epithelial cells may play a role in eosinophil recruitment
by releasing chemotactic activity in response to bleomycin.
The increased release of granulocyte-macrophage colonystimulating factor suggests a novel role for this cytokine in
pulmonary fibrosis. Further investigation is needed to
confirm the present results in primary cell culture or by
examination of tissues.
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