Smoke extract stimulates lung fibroblasts to release
neutrophil and monocyte chemotactic activities
Etsuro
Sato1,
Sekiya
Koyama1,
Akemi
Takamizawa1,
Takeshi
Masubuchi1,
Keishi
Kubo1,
Richard A.
Robbins2,
Sonoko
Nagai3, and
Takateru
Izumi3
1 The First Department of
Internal Medicine, Shinshu University School of Medicine, Matsumoto
390-8621; 3 Kyoto University Chest
Disease Research Institute, Kyoto 606-01, Japan; and
2 Overton Brooks Veterans
Affairs and Louisiana State University Medical Centers, Shreveport,
Louisiana 71101
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ABSTRACT |
Accumulation of monocytes and neutrophils and
fibrous distortion of the airway are characteristics of airway disease
secondary to smoking. The presence of inflammatory cells and fibrosis
correlate, and, therefore, we postulated that lung fibroblasts might
release chemotactic activity for neutrophils and monocytes in response to smoke extract. To test this hypothesis, human fetal lung (HFL1) fibroblasts were cultured, and the supernatant fluid was evaluated for
neutrophil (NCA) and monocyte (MCA) chemotactic activities with a blind
well chamber technique. HFL1 fibroblasts released chemotactic activity
in response to smoke extract in a dose- and time-dependent manner
(P < 0.05). Checkerboard analysis
showed that the activity was predominantly chemotactic. Partial
characterization of the released chemotactic activity revealed that the
activity was partly heat labile, trypsin sensitive, and ethyl acetate
extractable. Lipoxygenase inhibitors and cycloheximide inhibited the
release of both NCA and MCA. Molecular-sieve chromatography revealed
that NCA and MCA were heterogeneous. NCA was inhibited by anti-human interleukin (IL)-8 and anti-granulocyte colony-stimulating factor antibodies and a leukotriene (LT)
B4-receptor antagonist.
Anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) and
anti-monocyte chemoattractant protein (MCP)-1 antibodies and an
LTB4-receptor antagonist inhibited MCA. Immunoreactive IL-8, granulocyte colony-stimulating factor, GM-CSF, and MCP-1 significantly increased in culture supernatant fluid
in response to smoke extract. Finally, smoke extract augmented the
expression of mRNAs of IL-8, GM-CSF, and MCP-1. These data demonstrate
that lung fibroblasts release NCA and MCA in response to smoke extract
and suggest that lung fibroblasts may modulate the inflammatory cell
recruitment into the lung.
interleukin-8; granulocyte colony-stimulating factor; monocyte
chemoattractant protein-1; granulocyte-macrophage colony-stimulating
factor; fibrosis; chemotaxis; smoking
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INTRODUCTION |
THE ASSOCIATION between cigarette smoking and
bronchitis and emphysema is well established (1, 2, 29). Several
mechanisms may explain how smoking can cause lung inflammation and
subsequent disease. One theory in the pathogenesis of emphysema is an
abnormal balance between proteases and antiproteases and/or oxidants
and antioxidants in the lung (3, 15, 30). An increased number of
neutrophils and monocytes activated by cigarette smoke produces large
amounts of proteases and oxidants responsible for lung destruction (12,
33, 34). Senior et al. (28) reported that experimental emphysema was
induced in animals by intratracheal instillation of purified human
neutrophil elastase. Monocytes also contain an elastase (27), and this
protease, although differing from the neutrophil enzyme in amount and
subcellular localization, is antigenically and biochemically closely
related to the elastase of neutrophils. It is reported that macrophage
elastase is sufficient for the development of emphysema that results
from chronic inhalation of cigarette smoke in mice because macrophage
elastase-deficient mice did not develop emphysema (11).
In a clinical study (13), increased numbers of macrophages,
neutrophils, and eosinophils have been demonstrated in bronchoalveolar lavage fluid from smokers. Pulmonary tissue injury and dysfunction in
cigarette smokers have been correlated with the number and activity of
inflammatory cells (24). Although it has been proposed that an influx
of inflammatory cells may occur because of chemotactic factors
generated in the lung in response to smoke, including complement
activation and macrophage- and airway epithelial cell-derived chemotactic factors, the precise mechanism remains to be elucidated.
Fibroblasts are the major cells responsible for the synthesis of matrix
elements in soft connective tissue and constitute 35-40% of the
cell in the interstitium of the lung. Although fibroblasts remain
largely quiescent under normal conditions, they are activated to
proliferate and synthesize various cytokines during inflammation, suggesting that they may contribute to the pathophysiology of certain
lung diseases (8, 22). Consistent with this concept, peribronchial
fibrous tissue in cigarette smoke-induced bronchiolitis correlates with
the presence of inflammatory cells (21). Although chemotactic activity
derived from macrophages and airway epithelial cells may recruit
inflammatory cells from the interstitium to bronchial lumens or
alveolar spaces, the regulation of inflammatory cell traffic from the
vasculature to the interstitium is uncertain.
Based on the above, we hypothesized that smoke extract might stimulate
lung fibroblasts to release neutrophil (NCA) and monocyte (MCA)
chemotactic activities. The results demonstrate that human fetal lung
(HFL1) fibroblasts released NCA and MCA, which were heterogeneous and
at least partially attributable to interleukin (IL)-8, granulocyte
colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and monocyte chemoattractant protein (MCP)-1, in response to smoke extract. These data suggest that
fibroblasts may regulate inflammatory cell traffic from the vasculature
to the interstitium.
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MATERIALS AND METHODS |
HFL1 fibroblast cultures. HFL1
fibroblasts (diploid, passage 27)
were purchased from a commercial source (American Type Culture Collection, Manassas, VA). The cells were suspended at 1 × 106 cells/ml in Ham's F-12 medium
supplemented with penicillin (50 U/ml), streptomycin (50 µg/ml), Fungizone (2 µg/ml), and 10% fetal calf serum
(all from GIBCO BRL, Life Technologies, Grand Island, NY). HFL1
fibroblast suspensions were added to 30-mm-diameter tissue culture
dishes (Corning, Corning, NY) and cultured at 37°C in a 5%
CO2 atmosphere. After 4-6
days, the cells had reached confluence, and the culture medium was
replaced with 2 ml of medium supplemented as above and cultured for an
additional day.
Preparation of cigarette smoke
extract. Smoke extract was prepared with a modification
of the method of Carp and Janoff (6) and Janoff (14).
Briefly, two cigarettes without filters were combusted with a modified
syringe-driven apparatus. The smoke was bubbled through 50 ml of
Hanks' balanced salt solution (HBSS; GIBCO BRL). The resulting
suspension was adjusted to pH 7.4 with concentrated NaOH and filtered
through a 0.2-µm-pore filter (Lida Manufacturing, Kenosha, WI) to
remove bacteria and large particles. The smoke extract prepared by this
method contained bacterial endotoxin < 10 ng/ml in 10% smoke extract
solution. The resulting smoke extract was applied to HFL1 fibroblast
cultures within 30 min of preparation.
Exposure of HFL1 fibroblasts to smoke
extract. Culture medium was removed from the cells by
washing twice with serum-free Ham's F-12 medium, and the cells were
incubated in the presence and absence of smoke extract (0, 0.5, 1, 5, and 10%) for 12, 24, 48, and 72 h at 37°C in a humidified 5%
CO2 atmosphere. Smoke extract was
not cytotoxic as assessed by cell shape, cell detachment, and trypan
blue exclusion after 72 h at 10% concentration. The supernatant fluids
were harvested and stored at
80°C until assayed. At least
six HFL1 fibroblast culture supernatant fluids were harvested for each
experimental condition.
Measurement of NCA and MCA.
Neutrophils were purified from heparinized normal human blood with the
method of Böyum (5). Briefly, 15 ml of blood were obtained and
sedimented with 3% dextran in isotonic saline for 45 min to separate
white blood cells from 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, St. Louis, MO). Contaminating red blood cells were lysed with 0.1%
KHCO3 and 0.83%
NH4Cl. The suspension was
centrifuged at 400 g for 5 min and
washed three times with HBSS. The resulting cell pellet 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 a final concentration of 3 × 106 cells/ml.
Mononuclear cells for the chemotaxis assay were obtained from normal
human volunteers by Ficoll-Hypaque density centrifugation to separate
the red blood cells and neutrophils from the mononuclear cells. The
mononuclear cells were harvested at the interface and centrifuged at
400 g for 5 min and washed three times
with HBSS. The resulting cell pellet routinely consisted of ~30%
monocytes and 70% lymphocytes by morphology and esterase staining
(Sigma), with >98% viable cells. The cells were suspended in Gey's
balanced salt solution (GIBCO BRL) containing 2% BSA (Sigma) at pH 7.2 to a final concentration of 5 × 106 cells/ml.
Chemotaxis was performed in 48-well microchemotaxis chambers (Neuro
Probe, Cabin John, MD) as previously described (10). The bottom wells
of the chamber were filled with 25 µl of 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 monocytes was placed over the samples. The
silicon gasket and the upper pieces of the chamber were applied, and 50 µl of the cell suspension were placed into the upper wells. The chambers were incubated in 5% CO2
humidified air at 37°C for 30 min for neutrophil chemotaxis and 90 min for monocyte chemotaxis. Nonmigrated cells were wiped away from the
filter. The filter was immersed in methanol for 5 min, stained with a
modified Wright's stain, and mounted on a glass slide. Cells that had
completely migrated through the filter were counted with light
microscopy, and 10 random high-power fields (HPF; ×1,000)/well
were counted.
To ensure that monocytes and 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 directions (Sigma).
To determine whether the migration was due to movement along a
concentration gradient (chemotaxis) or to stimulation of random migration (chemokinesis), a checkerboard analysis was performed with
HFL1 fibroblast culture supernatant fluid obtained from cells cultured
for 72 h in the presence of 5% smoke extract (38). This was done by
placing various dilutions (1:1, 1:4, 1:16, 1:64, and 1:256) of the
culture supernatant fluid with target cells below and above the membrane.
Partial characterization of NCA and
MCA. Partial characterization of NCA and MCA released
from the HFL1 fibroblast cultures was performed with cultured
supernatant fluid harvested after 72 h of incubation with 5% smoke
extract. Sensitivity to proteases was tested by incubating the culture
supernatant fluid with trypsin (100 µg/ml; Sigma) for 30 min at
37°C followed by addition of a 1.5 M excess of soybean trypsin
inhibitor to terminate the proteolytic activity. Lipid solubility was
evaluated by ethyl acetate extraction. Both the extracted and
extractant fluids were evaluated for chemotactic activity. Heat
sensitivity was determined by heating the supernatant fluid to 98°C
for 15 min.
Molecular-sieve column chromatography.
To determine the approximate molecular mass of the released chemotactic
activity, molecular-sieve column chromatography was performed with a
Sepadex G-200 column (Pharmacia, Piscataway, NJ) at a flow rate of 6 ml/h on 5% smoke extract-stimulated culture supernatant fluid
harvested after 72 h of incubation. The culture supernatant fluid was
eluted from the column with PBS, and the fractions were evaluated for
NCA and MCA in duplicate.
Metabolic inhibitors of chemotactic
activity. The effects of the nonspecific lipoxygenase
inhibitors nordihydroguaiaretic acid (100 µM; Sigma) and
diethylcarbamazine (1 mM; Sigma) and the 5-lipoxygenase inhibitor
AA-861 (100 µM; Takeda Pharmaceutical, Tokyo, Japan) on the release
of chemotactic activity in response to 5% smoke extract were
evaluated. To assess the requirement for protein synthesis in the
release of chemotactic activity, cycloheximide (20 µg/ml; Sigma) was
added to some cultures.
Leukotriene B4- and platelet-activating
factor-receptor antagonists.
The release of NCA and MCA was attenuated by the 5-lipoxygenase
inhibitor, and NCA and MCA were ethyl acetate extractable. Therefore,
the leukotriene (LT) B4-receptor
antagonist ONO-4057 (ONO Pharmaceutical, Tokyo, Japan) and the
platelet-activating factor (PAF)-receptor antagonist TCV-309
(10
5 M; Takea
Pharmaceutical, Tokyo, Japan) were used to assess the involvement of
LTB4 and PAF in NCA and MCA.
Measurement of LTB4 and PAF.
The concentration of LTB4 in the
culture supernatant fluid was measured by RIA as previously described
(17). PAF was measured with a scintillation proximity assay (32).
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 method used for the measurement of
LTB4.
Measurement of IL-8, G-CSF, MCP-1, GM-CSF, regulated
on activation normal T cells expressed and secreted, and transforming growth factor-
. The concentrations of
IL-8, G-CSF, and MCP-1 were measured in culture supernatant fluid
harvested from cells cultured with 5% smoke extract after 72 h of
incubation with an ELISA (R&D Systems, Minneapolis, MN). The minimum
concentrations detected by these ELISAs were 10 pg/ml for IL-8, 31.3 pg/ml for MCP-1, and 0.31 ng/ml for transforming growth factor
(TGF)-
. GM-CSF and regulated on activation normal T cells expressed
and secreted (RANTES) kits were purchased from Amersham. The minimum concentrations detected with these methods were 2.0 pg/ml for GM-CSF
and 15.6 pg/ml for RANTES. G-CSF was assayed with a commercial kit
(Chugai Pharmaceutical, Tokyo, Japan), with a minimum detection limit
of 1.0 pg/ml.
Evaluation of IL-8, G-CSF, MCP-1, and GM-CSF mRNA
expression. The protein secretions of IL-8, MCP-1, and
GM-CSF were augmented by smoke extract. RT-PCR was used to evaluate
mRNA expression of IL-8, MCP-1, and GM-CSF in HFL1 fibroblast cultures
in response to 5% smoke extract after 72 h of incubation. Total RNA
was extracted from HLF1 fibroblast cultures as previously described
(7). One microgram of total RNA was reverse transcribed with a
commercially available kit (Promega, Madison, WI) and then amplified
for 32 cycles in a Perkin-Elmer model 480 thermal cycler (denaturation at 94°C for 45 s, primer annealing at 72°C for 45 s, and primer extension at 72°C for 7 min). The sequences used in the present study were GM-CSF forward, 5'-GGAGCATGTGAATGCCATC-3',
and reverse, 5'-ATCTGGGTTGCACAGGAAG-3'; IL-8 forward,
5'-TACTCCAAACCTTTCCAACCC-3', and reverse,
5'-AACTTCTCCACAACCCTCTG-3'; MCP-1 forward,
5'-CAGCCAGATGCAATCAATGC-3', and reverse,
5'-GTGGTCCATGGAATCCTGAA-3'; and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forward,
5'-ACCACAGTCCATGCCATCA-3', and reverse,
5'-TCCACCACCCTGTTGCTGTA-3'. Preliminary studies indicated that >32 cycles were subsaturating for the mRNA tested and thus were
appropriate for a comparison of the relative levels of mRNA between
groups. PCR products were separated by electrophoresis on a 3% agarose
gel and visualized by 32P
exposure. PCR band densities were determined by a NIH Image analytic
program (National Institutes of Health, Bethesda, MD) on unaltered,
computer-scanned images. GAPDH was used as a housekeeping gene for PCR,
which was measured in both normal and stimulated RNA samples at each
point. Integrated optical density measurement of 10 separate GAPDH
samples did not vary >10% from the mean integrated optical density,
which is an indication of the expected variation resulting from the
experimental technique.
Statistics. In experiments where multiple experiments were
performed, differences between groups were tested for significance with
a one-way analysis of variance, with Duncan's multiple range test
applied to data at specific time and dose points. In experiments where
single measurements were made, the differences between groups were
tested with Student's paired t-test.
In all cases, a P value of <0.05 was
considered significant. Data are expressed as means ± SE.
 |
RESULTS |
Release of NCA and MCA from HFL1 fibroblasts. HFL1
fibroblasts released NCA and MCA in a dose-dependent manner above a 1% concentration (P < 0.05; Fig.
1). HFL1 fibroblasts also released NCA and
MCA in a time-dependent manner after 48 h (Fig.
2). Smoke extract itself was not
chemotactic for neutrophils or monocytes (data not shown).

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Fig. 1.
Dose-dependent release of neutrophil
(A) and monocyte
(B) chemotactic activities in
response to smoke extract (n = 8 monolayers). * P < 0.05 compared with control culture supernatant fluid.
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Fig. 2.
Time-related release of neutrophil
(A) and monocyte
(B) chemotactic activities in
response to 5% smoke extract (n = 8 monolayers). , Chemotactic activity by cultures with smoke extract;
, unstimulated control cultures.
* P < 0.05 compared with 12-h
culture supernatant fluid.
# P < 0.05 compared with culture supernatant fluid without smoke
extract.
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The chemotactic response to LTB4
at a concentration of 10
7 M
was 1,020 ± 74 cells/10 HPF for neutrophils and 756 ± 34 cells/10 HPF for monocytes.
Checkerboard analysis revealed that the HFL1 fibroblast culture
supernatant fluid stimulated with smoke extract induced neutrophil and
monocyte migration with increasing concentrations in the presence of a
gradient (Table 1). Thus the migration of
neutrophils and monocytes was predominantly consistent with chemotactic
activity rather than chemokinetic activity.
Confirmation that the migrated cells were monocytes in the monocyte
chemotaxis assay was shown by several experiments. Greater than 90% of
the migrated cells were morphologically monocytes. Greater than 90% of
the migrated cells were esterase positive. 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. Both NCA and MCA were heterogeneous in character.
NCA and MCA both were partially sensitive to heat, ethyl acetate
extractable, and partially digested by trypsin (Fig.
3).

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Fig. 3.
Partial characterization of released neutrophil
(A) and monocyte
(B) chemotactic activities in
response to 5% smoke extract after 72 h of incubation
(n = 6 monolayers). EA, ethyl acetate;
RPMI, RPMI 1640 medium. * P < 0.05 compared with unstimulated human fetal lung (HFL1) fibroblast
culture supernatant fluid.
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Molecular-sieve column chromatography.
The released chemotactic activity obtained from HFL1 fibroblast culture
supernatant fluid incubated with 5% smoke extract for 72 h was
evaluated by molecular-sieve column chromatography with a Sephadex
G-200 column. These experiments revealed that the released NCA obtained
from unstimulated cells was heterogeneous in size (Fig.
4A).
At least four peaks of NCA that were just before BSA (molecular mass 66 kDa), before cytochrome c (molecular
mass 12.3 kDa), just after cytochrome
c, and near quinacrine (molecular mass
0.45 kDa) were separated by column chromatography. Stimulation with
smoke extract resulted in each of these peaks becoming more prominent.

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Fig. 4.
Molecular-sieve column chromatography of released neutrophil
(A) and monocyte
(B) chemotactic activities in
response to 5% smoke extract after 72 h of incubation. ,
Chemotactic activity obtained from HFL1 fibroblast culture supernatant
fluid stimulated with smoke extract; , chemotactic activity of
unstimulated HFL1 fibroblast culture supernatant fluid. Arrows,
molecular-mass markers.
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The released MCA from unstimulated and smoke extract-stimulated HFL1
fibroblast culture supernatant fluid was also heterogeneous and similar
to the released NCA (Fig. 4B). At
least four peaks of monocyte chemotactic activity that were just before
BSA, before cytochrome c, just after
cytochrome c, and near quinacrine were separated by column chromatography. Stimulation with smoke extract resulted in each of these peaks becoming more prominent.
Effects of metabolic inhibitors. The
culture supernatant fluid incubated with 5% smoke extract for 72 h and
with nordihydroguaiaretic acid, diethylcarbamazine, and AA-861 showed a
significant decrease in NCA and MCA (Fig.
5). Cycloheximide also significantly
inhibited the release of NCA and MCA
(P < 0.05; Fig. 5).

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Fig. 5.
Effect of nordihydroguaiaretic acid (NDGA), diethylcarbamazine (DEC),
AA-861, and cycloheximide (cyclo) on release of neutrophil
(A) and monocyte
(B) chemotactic activities in
response to 5% smoke extract after 72 h
(n = 8 monolayers).
* P < 0.05 compared with HFL1
fibroblast culture supernatant fluid stimulated with smoke extract.
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Effects of LTB4- and PAF-receptor
antagonists.
Both total NCA and MCA and the lowest-molecular-mass NCA and MCA
separated by column chromatography were significantly inhibited by
addition of the LTB4-receptor
antagonist ONO-4057 (Fig. 6). In contrast,
the effects of the PAF-receptor antagonist TCV-309 were not significant
for NCA and MCA. Each receptor antagonist at
10
5 M completely inhibited
the neutrophil migration to
10
7 M concentrations of
LTB4 and PAF, but the antagonists
showed no inhibitory effects on complement-activated serum-induced NCA or MCA (data not shown).

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Fig. 6.
Effect of leukotriene (LT) B4- and
platelet-activating factor (PAF)-receptor antagonists (ONO-4057 and
TCV-309, respectively) on released neutrophil
(A) and monocyte
(B) chemotactic activities in HFL1
fibroblast culture supernatant fluid in response to 5% smoke extract
after 72 h of incubation (n = 8 monolayers). * P < 0.05 compared with HFL1 fibroblast culture supernatant fluid stimulated with
smoke extract.
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Effects of smoke extract on the release of
LTB4.
The measurement of LTB4 by RIA
revealed that HFL1 fibroblasts released
LTB4 under basal unstimulated
conditions (278 ± 34 pg/ml). There was no significant increase in
LTB4 release from HFL1 fibroblasts
cultured with 5% smoke extract for 72 h (289 ± 29 pg/ml). PAF was
not detected in the unstimulated or smoke extract-stimulated culture
supernatant fluid after 72 h of incubation.
Effects of anti-IL-8, anti-G-CSF, anti-GM-CSF,
anti-MCP-1, anti-RANTES, and anti-TGF-
antibodies. Polyclonal blocking antibodies were
evaluated for their capacity to reduce NCA and MCA. Anti-IL-8 and
anti-G-CSF antibodies significantly reduced NCA. Anti-MCP-1 and
anti-GM-CSF antibodies significantly reduced MCA (Fig.
7). The effects of anti-IL-8, anti-G-CSF,
anti-GM-CSF, and anti-MCP-1 antibodies were also evaluated on the
column chromatography fractions. These antibodies reduced the
chemotactic activity at the corresponding molecular-mass
peak (data not shown). In contrast, anti-RANTES and anti-TGF-
antibodies did not block MCA.

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Fig. 7.
Effects of anti-interleukin (IL)-8 and anti-granulocyte
colony-stimulating factor (G-CSF) polyclonal antibodies on neutrophil
chemotactic activity (A) and of
anti-granulocyte-macrophage colony-stimulating factor (GM-CSF),
anti-transforming growth factor (TGF)- , anti-regulated on activation
normal T cells expressed and secreted (RANTES), and anti-monocyte
chemoattractant protein (MCP)-1 polyclonal antibodies on monocyte
chemotactic activity (B) in culture
supernatant fluid stimulated with 5% smoke extract for 72 h
(n = 6 monolayers).
* P < 0.05 compared with HFL1
fibroblast culture supernatant fluid stimulated with smoke extract.
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Effects of smoke extract on the release IL-8, G-CSF,
GM-CSF, MCP-1, RANTES, and TGF-
. The
concentrations of IL-8 and G-CSF significantly increased in the culture
supernatant fluid in response to smoke extract (Fig.
8). The concentrations of GM-CSF and MCP-1 were also significantly increased (Fig. 9).
HFL1 fibroblasts constitutively released TGF-
, but the concentration
was not increased by smoke extract stimulation. RANTES was undetectable
in both unstimulated and smoke-stimulated culture supernatant fluids.

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Fig. 8.
Release of IL-8 (A) and G-CSF
(B) in response to 5% smoke extract
after 72 h (n = 6 monolayers). cont,
Control. * P < 0.05 compared
to unstimulated HFL1 fibroblast culture supernatant fluid.
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Fig. 9.
Release of GM-CSF (A), MCP-1
(B), and TGF-
(C) in response to 5% smoke extract
after 72 h of incubation (n = 8 monolayers). * P < 0.05 compared with unstimulated HFL1 fibroblast culture supernatant fluid.
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Augmentation of IL-8, GM-CSF, and MCP-1 mRNA
expression by smoke extract. Smoke extract treatment of
HFL1 for 6 h resulted in the augmented expression of IL-8, GM- CSF, and
MCP-1 mRNAs (Fig. 10). Although we
evaluated the expression of G-CSF, we could not detect a clear band of
G-CSF mRNA after 40 cycles of amplification in both stimulated and
unstimulated samples (data not shown).

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Fig. 10.
Augmented expression of IL-8, GM-CSF, and MCP-1 mRNAs by 5% smoke
extract after 72 h of incubation in HFL1 fibroblast monolayers.
IL-8/GAPDH, GM-CSF/GAPDH, and MCP-1/GAPDH, ratios of expression of
IL-8, GM-CSF, and MCP-1, respectively, to that of
glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Data are
representative of 4 experiments.
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DISCUSSION |
In the present study, HFL1 fibroblasts released NCA and MCA in response
to smoke extract in a dose- and time-dependent manner. The released
activity was heterogeneous and chemotactic. Anti-IL-8 and anti-G-CSF
antibodies and an LTB4-receptor
antagonist inhibited NCA. Anti-MCP-1 and anti-GM-CSF antibodies and an
LTB4-receptor antagonist inhibited
MCA. In response to smoke extract, IL-8, G-CSF, MCP-1, and GM-CSF were
all increased in HFL1 fibroblast culture supernatant fluid. Finally,
the mRNA expression of IL-8, GM-CSF, and MCP-1 was augmented by smoke
extract in HFL1 fibroblast monolayers. These data suggest that
fibroblasts may play a role in the pathogenesis of smoking-induced lung
diseases by releasing NCA and MCA in response to cigarette smoke.
Inflammatory cells are present in increased numbers in the lungs of
smokers (13). Both in vitro and in vivo studies support the potential
for inflammatory cells to damage lung parenchyma. The alteration of
lung matrix could occur by a variety of mechanisms including the
release of oxygen radicals and proteases (14, 23). The present study
suggests that increased numbers of neutrophils and macrophages in the
lung secondary to smoking may be due, at least in part, to NCA and MCA
released from fibroblasts in response to smoke. Consistent with
this hypothesis, peribronchial fibrosis and inflammation
secondary to smoking often coexist (20, 31).
The present study demonstrates that several chemotactic factors were
released by fibroblasts that may contribute to inflammatory cell
recruitment. Partial characterization revealed that the released NCA
and MCA were partially ethyl acetate extractable. Molecular-sieve column chromatography showed a large chemotactic peak in the lowest molecular-mass range, and an
LTB4-receptor antagonist inhibited this chemotactic activity. Furthermore, the concentration of
LTB4 was high enough to produce
chemotactic activity (17, 19). Although smoke extract did not increase
the release of LTB4 into HFL1
fibroblast culture supernatant fluid,
LTB4 appeared to be one of the
predominant chemotactic factors released.
In contrast, the trypsin sensitivity of the chemotactic activity along
with the inhibition of the release of chemotactic activity by
cycloheximide suggests that chemotactic activity released from HFL1
fibroblasts is at least partially dependent on protein synthesis. Molecular-sieve column chromatography revealed increases in the higher-molecular-mass peaks of chemotactic activity in response to
smoke extract. The antibodies to IL-8, G-CSF, MCP-1, and GM-CSF inhibited NCA and MCA. Consistent with the inhibition of chemotactic activity by blocking antibodies, these cytokines were significantly increased in the culture supernatant fluid in response to smoke extract. The expression of these cytokine mRNAs was augmented by smoke
extract. These data suggest that these cytokines may play important
roles in the recruitment of inflammatory cells into the lungs of smokers.
Early descriptions of cytokines focused on their production by immune
and inflammatory effector cells. However, it is apparent that
structural cells are also capable of releasing many cytokines. Fibroblasts are known to produce a variety of cytokines including IL-8
(25), GM-CSF (25), TGF-
(16), and MCP-1 (26) in response to a
variety of stimuli. However, the relationship between smoking and the
release of these cytokines has not been established. The present study
demonstrated that HFL1 fibroblasts released these cytokines as
chemotactic factors in response to smoke extract and suggest that these
cytokines may play a role in smoking-induced lung disease by recruiting
inflammatory cells.
The release of NCA and MCA from human lung fibroblasts was a two- to
threefold increase from the constitutive release of NCA and MCA, and
the induction of fibroblast chemotactic activity by smoke extract was
modest compared with fractions isolated from unstimulated fibroblasts.
However, the released NCA and MCA from fibroblasts in response to smoke
extract was more than those from 106 alveolar macrophages/ml
stimulated with lipopolysaccharide (data not shown). Thus fibroblasts
may play an important role in the recruitment of inflammatory cells in
response to smoke extract.
Although TGF-
was detected in the cell culture supernatant fluid,
anti-TGF-
antibodies did not attenuate MCA. TGF-
induces monocyte
chemotaxis at concentrations of 0.1-10 pg/ml (35). At higher
concentrations, the chemotactic response of monocytes declines. TGF-
is usually released by cells in an inactive form (9, 36). The
biologically inactive form of TGF-
is unable to bind to its
receptor, and the release of inactive TGF-
may account for the lack
of inhibition of MCA in the HFL1 fibroblast culture supernatant fluid
by anti-TGF-
antibodies.
G-CSF has been reported to induce neutrophil migration at
concentrations > 10-100 U/ml (7-70 ng/ml) (37). The
concentration of G-CSF in the culture supernatant fluid was relatively
low in the present study. However, blocking anti-G-CSF antibodies
reduced NCA by up to 60%. Recently, Koyama et al. (18)
have found that 10-1,000 pg/ml of G-CSF induced significant NCA.
Although it is possible that G-CSF may be facilitating the chemotactic
response of other peptides (4), the concentration of G-CSF in the
culture supernatant fluid exceeded the lower chemotactic threshold
observed in our laboratory.
In conclusion, HFL1 fibroblasts release NCA and MCA in response to
smoke extract in vitro. The released activity was heterogeneous and
could be attributed to several cytokines. These data suggest that lung
fibroblasts may play an important role in inflammatory cell recruitment
in disorders associated with cigarette smoking.
 |
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 Department of Internal Medicine, Shishu Univ. School of
Medicine, 3-1-1 Ashai Matsumoto 390, Japan.
Received 4 August 1998; accepted in final form 30 July 1999.
 |
REFERENCES |
1.
Auerbach, O.,
E. C. Hammond,
L. Garfinkel,
and
C. Benante.
Relation of smoking and age to emphysema: whole lung section study.
N. Engl. J. Med.
286:
853-856,
1972[Medline].
2.
Auerbach, O.,
A. P. Stout,
E. C. Hammond,
and
L. Garfinkel.
Smoking habits and age in relation to pulmonary changes: rupture of alveolar septums, fibrosis and thickening of walls of small arteries and arterioles.
N. Engl. J. Med.
269:
1045-1054,
1963.
3.
Blue, M. L.,
and
A. Janoff.
Possible mechanisms of emphysema in cigarette smokers: release of elastase from human polymorphonuclear leukocytes by cigarette smoke condensate in vitro.
Am. Rev. Respir. Dis.
117:
317-325,
1978[Medline].
4.
Bober, L. A.,
M. J. Grace,
C. Pugliese-Sivo,
A. Rojas-Triana,
T. Waters,
L. M. Sullivan,
and
S. K. Narula.
The effect of GM-CSF and G-CSF on human neutrophil function.
Immunopharmacology
29:
111-119,
1995[Medline].
5.
Böyum, A.
Isolation of mononuclear cells and granulocytes from human blood.
Scand. J. Clin. Lab. Invest.
97:
77-89,
1968.
6.
Carp, H.,
and
A. Janoff.
A possible mechanism of emphysema in smokers. In vitro suppression of serum elastase-inhibitory capacity by fresh cigarette smoke and its prevention by antioxidants.
Am. Rev. Respir. Dis.
118:
617-621,
1978[Medline].
7.
Chomczynski, P.,
and
N. Sacchi.
Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction.
Anal. Biochem.
162:
156-159,
1987[Medline].
8.
Clark, R. A. F.,
and
P. M. Henson.
The Molecular and Cellular Biology of Wound Healing. New York: Plenum, 1995.
9.
Harpel, J. G.,
N. C. Metz,
S. Kojima,
and
D. B. Rifkin.
Control of transforming growth factor-
1 activity: latency vs. activation.
Prog. Growth Factor Res.
4:
321-335,
1992[Medline].
10.
Harvath, L.,
W. Falk,
and
E. J. Leonard.
Rapid quantification of neutrophil chemotaxis: use of polyvinylpyrrolidone-free polycarbonate membrane in a multiwell assembly.
J. Immunol. Methods
37:
39-45,
1980[Medline].
11.
Hautamaki, R. D.,
D. K. Kobayashi,
R. M. Senior,
and
S. D. Shapiro.
Requirement for macrophage elastase for cigarette smoke-induced emphysema in mice.
Science
277:
2002-2004,
1997[Abstract/Free Full Text].
12.
Hinman, L. M.,
C. A. Stevens,
R. A. Matthay,
and
J. B. Gee.
Elastase and lysozyme activities in human alveolar macrophages. Effects of cigarette smoking.
Am. Rev. Respir. Dis.
121:
263-271,
1980[Medline].
13.
Hunninghake, G. W.,
and
R. G. Crystal.
Cigarette smoking and lung destruction: accumulation of neutrophils in the lungs of cigarette smokers.
Am. Rev. Respir. Dis.
128:
833-838,
1983[Medline].
14.
Janoff, A.
Elastases and emphysema. Current assessment of the protease-antiprotease hypothesis.
Am. Rev. Respir. Dis.
132:
417-433,
1985[Medline].
15.
Janoff, A.,
and
H. Carp.
Possible mechanisms of emphysema in smokers: cigarette smoke condensate suppresses protease inhibition in vitro.
Am. Rev. Respir. Dis.
116:
65-72,
1977[Medline].
16.
Kelley, J.,
J. P. Fabisiak,
K. Hawes,
and
M. Absher.
Cytokine signaling in the lung: transforming growth factor-
secretion by lung fibroblasts.
Am. J. Physiol.
260 (Lung Cell. Mol. Physiol. 4):
L123-L128,
1991[Abstract/Free Full Text].
17.
Kishikawa, K.,
N. Tateishi,
T. Maruyama,
R. Seo,
M. Toda,
and
T. Miyamoto.
ONO-4057, a novel, orally active leukotriene B4 antagonist: effects on LTB4-induced neutrophil functions.
Prostaglandins
44:
261-275,
1992[Medline].
18.
Koyama, S.,
E. Sato,
T. Masubuchi,
A. Takamizawa,
K. Kubo,
S. Nagai,
and
T. Izumi.
Human lung fibroblasts release chemokinetic activity for monocytes constitutively.
Am. J. Physiol.
275 (Lung Cell. Mol. Physiol. 19):
L223-L231,
1998[Abstract/Free Full Text].
19.
Koyama, S.,
E. Sato,
T. Masubuchi,
A. Takamizawa,
K. Kubo,
S. Nagai,
and
T. Izumi.
Alveolar type II-like cells release G-CSF as neutrophil chemotactic activity.
Am. J. Physiol.
275 (Lung Cell. Mol. Physiol. 19):
L687-L695,
1998[Abstract/Free Full Text].
20.
McLean, K. H.
The pathogenesis of pulmonary emphysema.
Am. J. Med.
25:
62-74,
1958.
21.
Petty, T. L.,
W. Silers,
and
R. E. Stanford.
Radial traction and small airways disease in excised human lungs.
Am. Rev. Respir. Dis.
133:
132-135,
1986[Medline].
22.
Raghu, G.,
and
T. Kvanagh.
The human lung fibroblast: a multifaceted target and effector cell.
In: Interstitial Pulmonary Diseases, edited by R. Barrios,
and M. Selman. Boca Raton, FL: CRC, 1991, p. 134.
23.
Repine, J. E.,
A. Bast,
and
I. Lankhorst.
Oxidative stress in chronic obstructive pulmonary disease. Oxidative Stress Study Group.
Am. J. Respir. Crit. Care Med.
156:
341-357,
1997[Free Full Text].
24.
Richards, G. A.,
A. J. Theron,
C. A. Van de Merwe,
and
R. Anderson.
Spirometric abnormalities in young smokers correlate with increased chemiluminescence responses of activated blood phagocytes.
Am. Rev. Respir. Dis.
139:
181-187,
1989[Medline].
25.
Rolfe, M. W.,
S. L. Kunkel,
T. J. Standiford,
S. W. Chensue,
R. M. Allen,
H. L. Evanoff,
S. H. Phan,
and
R. M. Streiter.
Pulmonary fibroblast expression of interleukin-8: a model for alveolar macrophage-derived cytokine networking.
Am. J. Respir. Cell Mol. Biol.
5:
493-501,
1991[Medline].
26.
Rolfe, M. W.,
S. L. Kunkel,
T. J. Standiford,
M. B. Orringer,
S. H. Phan,
H. L. Evanoff,
M. D. Burdick,
and
R. M. Streiter.
Expression and regulation of human pulmonary fibroblast-derived monocyte chemotactic peptide-1.
Am. J. Physiol.
263 (Lung Cell. Mol. Physiol. 7):
L536-L545,
1992[Abstract/Free Full Text].
27.
Senior, R. M.,
E. J. Campbell,
J. A. Landis,
F. R. Cox,
C. Kuhn,
and
H. S. Koren.
Elastase of U-937 monocytelike cells: comparisons with elastase derived from human monocytes and neutrophils and murine macrophagelike cells.
J. Clin. Invest.
69:
384-393,
1982[Medline].
28.
Senior, R. M.,
H. Tegner,
C. Kuhn,
K. Ohlsson,
B. C. Starcher,
and
J. A. Pierce.
The induction of pulmonary emphysema with human leukocyte elastase.
Am. Rev. Respir. Dis.
116:
469-475,
1977[Medline].
29.
Snider, G. L.
The pathogenesis of emphysema
twenty years of progress.
Am. Rev. Respir. Dis.
124:
321-324,
1981[Medline].
30.
Snider, G. L.,
E. C. Lucey,
and
P. J. Stone.
Animal models of emphysema.
Am. Rev. Respir. Dis.
133:
149-169,
1986[Medline].
31.
Spain, D. M.,
and
G. Kaufman.
The basic lesion in chronic pulmonary emphysema.
Am. Rev. Respir. Dis.
68:
24-30,
1953.
32.
Terashita, Z.,
M. Kawamura,
M. Takatani,
S. Tsushima,
Y. Imura,
and
N. Nishikawa.
Beneficial effects of TCV-309, a novel potent and selective platelet activating factor antagonist in endotoxin and anaphylactic shock in rodents.
J. Pharmacol. Exp. Ther.
260:
748-755,
1992[Abstract].
33.
Thompson, A. B.,
D. Daughton,
R. A. Robbins,
M. Ghafouri,
M. Oehlerking,
and
S. I. Rennard.
Intraluminal airway inflammation in chronic bronchitis: characterization and correlation with clinical parameters.
Am. Rev. Respir. Dis.
140:
1527-1537,
1989[Medline].
34.
Thompson, A. B.,
G. Heurta,
R. A. Robbins,
J. H. Sisson,
J. R. Spurzem,
S. Von Essen,
K. A. Rickard,
D. J. Romberger,
I. Rubinstein,
J. P. Metcalf,
R. S. Yates,
M. Ghafouri,
M. G. Stahl,
D. M. Daughton,
and
S. I. Rennard.
The bronchitis index. A semiquantitative visual scale for the assessment of airways inflammation.
Chest
103:
1482-1488,
1993[Abstract].
35.
Wahl, S. M.,
D. A. Hunt,
L. M. Wakefield,
N. McCartney-Francis,
L. M. Wahl,
A. B. Roberts,
and
M. R. Sporn.
Transforming growth factor type
induces monocyte chemotaxis and growth factor production.
Proc. Natl. Acad. Sci. USA
84:
5788-5792,
1987[Abstract].
36.
Wakefield, L. M.,
D. M. Smith,
T. Masui,
C. C. Harris,
and
M. B. Sporn.
Distribution and modulation of the cellular receptor for transforming growth factor-beta.
J. Cell Biol.
105:
965-975,
1987[Abstract].
37.
Wang, J. M.,
Z. G. Chen,
S. Collela,
M. A. Bonilla,
K. Welte,
C. Bordignon,
and
A. Mantovani.
Chemotactic activity of recombinant human granulocyte colony-stimulating factor.
Blood
72:
1456-1460,
1988[Abstract].
38.
Zigmond, S. H.,
and
J. G. Hirsch.
Leukocyte locomotion and chemotaxis: new methods for evaluation and demonstration of a cell-derived chemotactic factor.
J. Exp. Med.
137:
387-410,
1973[Medline].
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