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1 The First Department of Internal Medicine, We evaluated the potential of
A549 cells, an alveolar type II epithelial cell line, to release
granulocyte colony-stimulating factor (G-CSF), in addition to
interleukin (IL)-8 and leukotriene B4, as neutrophil
chemotactic activity (NCA). Human recombinant IL-1
granulocyte colony-stimulating factor; type II pneumocyte; neutrophil chemotaxis
SEQUESTRATION of peripheral blood neutrophils within
the lung is characteristic of a number of acute pulmonary infections and lung injury (10, 11, 29). The presence of neutrophils is determined
by the expression of adhesion molecules and local generation of
chemotactic agents, which direct neutrophil migration from the vascular
compartment to the alveolar space along chemotactic gradients.
Substantial investigations have focused on alveolar macrophages as a
primary source of chemotactic factors and chemokines (9, 17, 20).
However, neutrophil chemotactic activity (NCA) has been reported to be
produced by endothelial cells (24), fibroblasts (25), and alveolar and
airway epithelial cells (13, 19, 22).
Alveolar type II epithelial cells (ATII cells) have been shown to play
a key role in the regulation of the alveolar space. ATII cells
synthesize and secrete surfactant, control the volume and composition
of the epithelial lining fluid, and proliferate and differentiate into
type I alveolar epithelial cells after lung injury to maintain the
integrity of the alveolar wall (16). ATII cells have a role in
modulating immunologic activity in the alveolar space. Both in vivo and
in vitro data suggest that ATII cells could participate in the
intra-alveolar cytokine network by secreting interleukin (IL)-8 (11,
22), IL-6 (6), monocyte chemoattractant protein (MCP)-1 (23), regulated
on activation normal T cells expressed and secreted (RANTES),
granulocyte-macrophage colony-stimulating factor (GM-CSF), transforming
growth factor (TGF)- Granulocyte colony-stimulating factor (G-CSF) plays significant roles
in neutrophil migration (27), activation (26), and survival (5). G-CSF
has been reported to be produced from macrophages, lymphocytes (4, 18),
fibroblasts (2), and endothelial cells (12) in response to certain
stimuli. However, the potential of ATII cells to produce G-CSF is
uncertain, and the regulation of G-CSF release as NCA from ATII cells
is undetermined. In the present study, we demonstrated that A549 cells
released G-CSF as NCA in response to IL-1 Culture and identification of ATII cells.
Because of difficulty in obtaining primary human type II epithelial
cells of sufficient purity, A549 cells (American Type Culture
Collection, Rockville, MD), an alveolar type II cell line derived from
an individual with alveolar carcinoma (26), were used. These cells
retained many of the characteristics of normal type II cells, such as
surfactant protein, cytoplasmic multilamellar inclusion bodies, and
cuboidal appearance, and have been extensively used to assess type II
pneumocyte effector cell function (6, 14, 15, 21-23). A549 cells
were grown as a monolayer on 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; GIBCO, Grand
Island, NY), streptomycin (50 µg/ml; GIBCO), Fungizone (2 µg/ml;
GIBCO), and 10% heat-inactivated FCS (GIBCO). The cells from a
monolayer 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 1.0 × 106 cells/ml in 35-mm tissue
culture dishes. The cells were grown to confluence in the dish for
5-7 days. After the cells reached confluence, they were used for
experiments.
Exposure of A549 cells to a variety of stimuli.
Serum-containing medium was removed from the cells by washing them
twice with serum-free Ham's F-12 medium; and then the cells were
incubated with Ham's F-12 medium without FCS in the absence and
presence of human recombinant IL-1
ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References
stimulated A549
cells to release NCA in a time- and dose-dependent fashion. The
released NCA was blocked by mouse anti-human G-CSF polyclonal antibody.
Molecular-sieve column chromatography revealed that IL-1 induced the
release of a 19- to 20-kDa chemotactic mass that was inhibited by
anti-human G-CSF antibody. IL-1 stimulated the release of G-CSF in a
dose-dependent fashion, but the time-dependent profile of G-CSF showed
that the concentration of G-CSF declined after 48 h. Tumor necrosis
factor (TNF)-
, Escherichia coli lipopolysaccharide (LPS),
and bradykinin (BK) stimulated A549 cells to release NCA that was
inhibited by anti-G-CSF antibody. The release of G-CSF in response to
TNF-, LPS, and BK was significantly increased. The similar
concentrations of human recombinant G-CSF (10-1,000 pg/ml) as in
the supernatant fluid induced neutrophil chemotaxis. G-CSF mRNA was
expressed time and dose dependently at 4 h and declined after 4 h in
response to IL-1 as evaluated by RT-PCR. The expression of G-CSF
mRNA was also observed by TNF-, LPS, and BK stimulation. These data
suggest that type II alveolar epithelial cells may produce G-CSF as NCA
and may participate in the regulation of leukocyte extravasation.
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
constitutively and in response to tumor
necrosis factor (TNF)- and IL-1 (14, 15).
, TNF-,
lipopolysaccharide (LPS), and bradykinin (BK). The expression of G-CSF
mRNA was augmented in response to each stimulus. These data suggest
that ATII cells may produce G-CSF as NCA and may participate in the
regulation of neutrophil extravasation into the lung.
METHODS
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Abstract
Introduction
Methods
Results
Discussion
References
(500, 50, 5, and 0.5 pg/ml; Genzyme, Cambridge, MA), human recombinant TNF- (1,000 U/ml; Genzyme), human recombinant interferon (IFN)-
(500 U/ml; Genzyme), Escherichia coli LPS (serotype 0127:8; Difco, Detroit, MI),
BK (100 µM; Sigma, St. Louis, MO), histamine (100 µM; Sigma), and serotonin (100 µM; Sigma) and cultured for 12, 24, 48, and 72 h; the
supernatant fluids were evaluated for neutrophil chemotaxis and G-CSF
concentration.
(500 pg/ml) for 2, 4, 8, and 12 h, washed with Hanks' balanced salt
solution (GIBCO), and evaluated by RT-PCR. Because G-CSF mRNA was
expressed most intensely after 4 h of exposure to IL-1, dose-dependent expression was evaluated at IL-1 concentrations of
500, 50, and 5 pg/ml. For TNF- (1,000 U/ml), LPS (100 µg/ml), and
BK (100 µM) stimulation, A549 cells were treated for 4 h, and then
mRNA evaluation was performed.
Measurement of NCA. Polymorphonuclear leukocytes were purified from heparinized normal human blood by the method of Boyum (1). Briefly, 15 ml of venous heparinized blood were suspended in the same volume of 3% dextran (Sigma) in isotonic saline for 30 min. The neutrophil-rich upper layer was aspirated and centrifuged at 400 g for 5 min, and the cell pellet was resuspended in lysing solution consisting of 0.1% KHCO3 and 0.83% NH4Cl. The suspension was then centrifuged and washed three times in Hanks' balanced salt solution. The viability of recovered neutrophils was >98% as assessed by trypan blue and erythrosin exclusion. The cells were suspended in Gey's balanced salt solution (GIBCO) containing 2% BSA (Sigma) at pH 7.2 to give a final concentration of 3.0 × 106 cells/ml.
The chemotaxis assay was performed in 48-well microchemotaxis chambers (Neuroprobe, Cabin John, MD) as previously described (8). The bottom wells of the chambers were filled with 25 µl of fluid containing the chemotactic stimulus. Each sample was tested in duplicate. A polycarbonate filter (Nuclepore, Pleasanton, CA) with a pore size of 3 µm for neutrophil chemotaxis was placed over the bottom wells. The silicon gasket and upper pieces of the chamber were applied, and the entire assembly was preincubated at 37°C in humidified air for 15 min before the upper wells were filled with 50 µl of cell suspension. The chamber was incubated in humidified 5% CO2 at 37°C for 30 min. The chamber was disassembled after the incubation, and the filter was fixed, stained with Diff-Quik (American Scientific Products, McGaw Park, IL), and mounted on a glass slide. Cells that completely migrated through the filter were counted in 5 random high-power fields (HPF; ×1,000) from each duplicate well. Chemotactic response was defined as the mean number of migrated cells per HPF. Ham's F-12 medium without FCS was incubated identically with A549 cells, and the supernatant fluids harvested were used to determine background neutrophil migration. Formyl-methionyl-leucyl-phenylalanine (f MLP; 10
8 M in Ham's F-12 medium; Sigma) and normal human
serum, which was complement activated by incubation with E. coli LPS 0127:B8 and diluted 10-fold with Ham's F-12 medium, were
used as positive controls (13).
Effects of polyclonal antibodies to G-CSF and IL-8 of leukotriene and B4-receptor antagonist on NCA. The neutralizing polyclonal antibodies to G-CSF and IL-8 were purchased from Genzyme. The leukotriene (LT) B4-receptor antagonist ONO-4057 was a kind gift from ONO Pharmaceutical (Tokyo, Japan).
Specificity of polyclonal rabbit anti-human G-CSF has been shown to bind to G-CSF by ELISA and dot analysis. There was no detectable cross-reactivity with human IL-3, monocyte CSF (M-CSF), or GM-CSF. Neutralizing activity was assessed by human G-CSF bioactivity by inhibiting 32D cell proliferation. However, cross-reactivity with G-CSF from species other than humans has not been tested. The IL-8 antibody recognized 77-, 72-, and 69-amino acid forms of human IL-8. No cross-reactivity was observed with human growth-related protein-, rat cytokine-induced neutrophil chemoattractant, human
macrophage inflammatory protein (MIP)-1, MIP-1, human MCP-1,
human MCP-2, f MLP, LTB4, or human C5a. Neutralization of IL-8 activity was proved by inhibiting IL-8-induced chemotaxis in a
Boyden chamber assay. Species specificity showed that, in vitro, this
antibody recognized IL-8 from rhesus macaques and an IL-8-like product
in rats and weakly recognized pig IL-8. Cross-reactivity with other
species has not been tested.
The antibodies inhibited the chemotactic activity of purified G-CSF at
concentrations of 500-5,000 pg/ml and of IL-8 at concentrations of
1-20 ng/ml, which were relevant to the concentrations in the present study.
G-CSF and IL-8 antibodies and ONO-4057 (10 µM) were added to A549
cell supernatant fluids to inhibit the effects of G-CSF, IL-8, and
LTB4 and incubated for 30 min in 37°C. Then these samples were used for chemotactic assay. These antibodies and antagonist did
not influence the neutrophil chemotactic response to
endotoxin-activated serum and f MLP (data not shown).
Partial purification of G-CSF by molecular-sieve column
chromatography.
To determine the molecular weight of NCA in the A549 cell supernatant
fluids, which were harvested after 48 h of incubation in response to
IL-1, TNF-, LPS, and BK, molecular-sieve column chromatography
was performed by using Sephadex G-100 (25 × 1.25 cm; Pharmacia,
Piscataway, NJ) at a flow rate of 6 ml/h. The A549 cell supernatant
fluid was eluted with PBS, and every fraction after the void volume was
evaluated for NCA in duplicate. The neutrophil chemotactic peak at a
molecular mass of 19-20 kDa was also treated with anti-G-CSF
antibody, and NCA was evaluated.
Measurement of G-CSF and IL-8 in the supernatant fluids. The concentrations of G-CSF and IL-8 in the A549 cell supernatant fluids stimulated by a variety of stimuli were measured by ELISA according to the manufacturer's direction. G-CSF ELISA kit was purchased from Amersham, and the minimum concentration of G-CSF detected by this method was 31.9 pg/ml. IL-8 kit was obtained from R&D Systems (Minneapolis, MN), and the minimum concentration of detectable IL-8 was 31.3 pg/ml.
RT-PCR.
RT-PCR was used to detect mRNA for G-CSF synthesis by A549 cells. Total
RNA was extracted from A549 cells as previously described (3). One
microgram of total RNA was reverse transcribed into cDNA with a cDNA
synthesis kit (Boehringer Mannheim, Mannheim, Germany) and then
amplified with Taq DNA polymerase (Boehringer Mannheim) for
27 cycles in a Perkin-Elmer Gene Amp PCR System 9600 (denaturation at
94°C for 30 s, primer annealing at 55°C for 30 s, and primer
extension at 72°C for 30 s). The G-CSF sense, antisense, and probe
used in the present study were 5'-GCTTAGAGCAAGTGAGGAAG-3', 5'-AGGTGGCGTAGAACGCGGTA-3', and 5'-ACCCAGGGTGCCATGCCGGCCTTCGCCTCT-3', respectively. Preliminary studies indicated that 27 cycles were subsaturating for mRNA tested and were thus appropriate for comparison of relative levels of mRNA between groups. PCR products were separated by electrophoresis on a 3% agarose gel and were visualized by labeled
32P exposure. PCR band densities were determined by the NIH
Image analytic program (National Institutes of Health, Bethesda, MD) on
unaltered, computer-scanned images. -Actin mRNA, which has been
shown not to change by stimulation, was measured in both normal and
stimulated RNA samples at each point with the same cDNA that was
analyzed for cytokines (data not shown). Integrated optical density
measurements of 10 separate -actin samples did not vary by >33%
from the mean integrated, optical density, which is an indication of
the expected variation resulting from the experimental technique.
Statistics. 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 |
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Abstract
Introduction
Methods
Results
Discussion
References
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Release of NCA from A549 cell monolayers.
A549 cells released NCA in response to IL-1 in a dose-related
fashion (P < 0.001; Fig. 1).
The lowest dose of IL-1 to stimulate A549 cells was 5.0 pg/ml.
Increasing concentrations of IL-1 up to 500 pg/ml progressively
increased the release of NCA. The release of NCA began 12 h of exposure
to IL-1, and the released activity reached the plateau at 48 h (Fig.
2). TNF-, LPS, BK, and IFN- also
induced the release of NCA from A549 cells in a time- and dose-dependent fashion (data not shown). However, the releasing potential of NCA by histamine and serotonin was weak and not
significant compared with control values. The chemotactic activities in
response to f MLP and activated serum were 70.4 ± 8.7 and 85.6 ± 15.3 neutrophils/HPF, respectively. IL-1 by itself in
the culture medium without cells and incubated identically did not show
significant chemotactic activity for neutrophils (data not shown).
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Inhibition of NCA by polyclonal antibodies to G-CSF and IL-8 and by
LTB4-receptor antagonist.
The neutralizing antibodies to G-CSF and IL-8 added to the A549 cell
supernatant fluids, which were harvested after 48 h of incubation in
response to IL-1, at the suggested concentrations inhibited NCA (30 and 35%, respectively). The LTB4-receptor antagonist also
inhibited NCA by 40% (Fig. 3). The
treatment with G-CSF antibody significantly inhibited NCA induced by
TNF-, LPS, and BK stimulation (Table 1).
The inhibition was ~40-50%. However, NCA induced by IFN- was
not inhibited by G-CSF antibody. The combined use of IL-8 and G-CSF
antibodies and the LTB4-receptor antagonist reduced NCA
(48.7 ± 4.3 to 18.7 ± 3.4 cells/HPF) but did not completely inhibit NCA.
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Partial purification of G-CSF by molecular-sieve column
chromatography.
Molecular-sieve column chromatography revealed that NCA was
heterogeneous in its size. Three peaks of chemotactic activity were
observed in IL-1-stimulated A459 cell supernatant fluids (Fig.
4). The molecular-mass chemotactic peak at
19-20 kDa was inhibited by the addition of anti-G-CSF polyclonal
antibody (28.5 ± 4.5 vs. 12.4 ± 2.5 cells/HPF;
P < 0.01; n = 4 monolayers).
The second molecular-mass peak was inhibited by IL-8 antibody
(31.5 ± 3.1 vs. 14.3 ± 2.3 cells/HPF; P < 0.01;
n = 4 monolayers), and the lowest-molecular-mass peak was
inhibited by the LTB4-receptor antagonist (34.3 ± 2.1
vs. 13.5 ± 1.8 cells/HPF; P < 0.01;
n = 4 monolayers). TNF-, LPS, and BK also induced three
neutrophil chemotactic peaks at similar molecular masses. The molecular
mass at 19-20 kDa in response to TNF-, LPS, and BK was
inhibited by anti-G-CSF antibody (Table 2).
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Measurement of G-CSF and IL-8 in the supernatant fluids.
The concentrations of G-CSF and IL-8 in A549 cell supernatant fluids in
response to IL-1 were increased in a dose-dependent fashion (Fig.
5). The release of IL-8 was observed at the
lower concentration of IL-1. The concentration of IL-8 increased
time dependently (Fig. 6B).
However, the concentration of G-CSF declined after 48 h of incubation
(Fig. 6A). TNF-, LPS, BK, IFN-, histamine, and
serotonin induced the release of IL-8 from A549 cells (Table 3). However, IFN-, histamine, and
serotonin did not stimulate A549 cells to release detectable amounts of
G-CSF.
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Neutrophil migration induced by human recombinant G-CSF. Neutrophil chemotaxis assay to human recombinant G-CSF (Kirin Pharmaceutical, Tokyo, Japan) was performed at concentrations ranging from 1 pg/ml to 10 ng/ml. Human recombinant G-CSF at 10-1,000 pg/ml induced neutrophil migration in a dose-dependent manner and declined thereafter. The concentration of G-CSF that induced maximum neutrophil chemotaxis was 100 pg/ml (Fig. 7).
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Induction of G-CSF mRNA by IL-1,
TNF-, BK, and LPS.
IL-1 at a concentration of 500 pg/ml induced the gene expression of
G-CSF in a time-dependent manner (Fig. 8).
The maximum expression of G-CSF mRNA was after 4 h of incubation, and
then it declined. IL-1 induced G-CSF mRNA expression in a
dose-dependent manner at 4 h (Fig. 9).
TNF-, LPS, and BK slightly induced G-CSF mRNA expression after 4 h
of incubation (Fig. 9).
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DISCUSSION |
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Abstract
Introduction
Methods
Results
Discussion
References
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In the present investigation, we evaluated the potential of IL-1 to
induce the release of G-CSF as NCA from A549 cells. IL-1 significantly stimulated A549 cells to release G-CSF in a dose- and
time-dependent manner. Molecular-sieve chromatography showed that
IL-1 induced three molecular masses (19-20 kDa, 8 kDa, and 400 Da) for NCA. Polyclonal blocking antibody to G-CSF significantly inhibited the chemotactic response in both crude and
column-fractionated supernatants. The release of G-CSF was
significantly augmented in response to IL-1. TNF-, LPS, and BK
also induced the release of G-CSF as NCA. The G-CSF mRNA evaluated by
RT-PCR showed a dose dependency at 4 h and declined after 4 h in
response to IL-1. The expression of G-CSF mRNA was also observed
with TNF-, LPS, and BK stimulation. These data suggest that ATII
cells may produce G-CSF as NCA in response to IL-1, TNF-, LPS,
and BK and may participate in the regulation of leukocyte
extravasation.
The characterization of released NCA in response to IL-1 is not
complete in the present study because the blocking antibody to G-CSF
attenuated the chemotactic activity up to 30% in response to IL-1.
Anti-G-CSF antibody inhibited NCA up to 40-50% in response to
TNF-, LPS, and BK. The involvement of IL-8 and LTB4 as
NCA was also significant. But the combined use of G-CSF and IL-8
antibodies and the LTB4-receptor antagonist did not
completely inhibit NCA. Because MIP-1 antibody did not influence NCA
and MIP-1 was not in the supernatant fluids (data not shown), the
involvement of MIP-1 was small. The possible candidates for NCA may
involve complements, 12-hydroxyeicosatetraenoic acid (12-HETE), and
15-HETE. Although complements and 12- and 15-HETE may be involved in
NCA released from A549 cells, IL-8, G-CSF, and LTB4
explained 80-90% of NCA released from A549 cells in response to
IL-1. Thus we speculate that the predominant NCA released from A549
cells was IL-8, G-CSF, and LTB4.
The dose response for IL-1 in releasing G-CSF and NCA did not appear
to agree perfectly. It has been reported that A549 cells released
predominantly IL-8 as NCA in response to TNF-, IL-1, and asbestos
(21, 22). The release of IL-8 was observed at the lower concentration
after a short time of exposure. The released IL-8 by a lower IL-1
concentration reached the concentration of neutrophil chemotaxis. This
was coincident with the previous report of IL-8 release from A549 cells
in response to 20 ng/ml of IL-1. The release of IL-8 was observed in
response to a variety of stimuli, including IFN-, histamine, and
serotonin. In contrast, the release of G-CSF needed a higher
concentration of IL-1 and specific stimuli. The release of
LTB4 in response to IL-1 was not different from that of
the unstimulated A549 cells (data not shown). The releasing pattern of
LTB4 was predominant at 24 h and then gradually increased.
Thus the relevant role of these chemotactic factors may be dependent on
the concentration of IL-1 or stimuli; i.e., at the lower
concentration of IL-1, the predominant NCA may be IL-8 and
LTB4 rather than G-CSF. However, the concentration of
IL-1 in the bronchoalveolar lavage fluid was fairly high. Thus G-CSF
may play an important role in neutrophil recruitment in lung
inflammation.
Both in vivo and in vitro data suggest that ATII cells could
participate in the intra-alveolar cytokine network by secreting IL-8
(21, 22), IL-6 (6), MCP-1 (23), RANTES, GM-CSF, and TGF- (14, 15)
constitutively and in response to TNF- and IL-1. In the present
study, we illustrated that A549 cells produced G-CSF. Because G-CSF
plays significant roles in neutrophil migration (27), activation (26),
and survival (5), the production of G-CSF from A549 cells may suggest
the amplification of the inflammatory responses of the lung by type II
epithelial cells in addition to the modulation of immunologic activity
in the alveolar space.
Locally produced chemoattractants are likely to play an important role in the regulation of neutrophil extravasation and localization. CSFs, including G-CSF, GM-CSF, and M-CSF, can influence the migratory capacity of leukocytes, although somewhat conflicting results have been obtained (7, 28, 30). GM-CSF has been reported to inhibit the migration of leukocytes in an agarose assay (7, 30). GM-CSF and M-CSF, on the other hand, act as relatively potent chemoattractants for neutrophils and monocytes in Boyden chambers (28). The apparent conflict between reports on the influence of GM-CSF on chemotaxis might be explained by the different exposure conditions. Wang et al. (27) reported that G-CSF induced migration of neutrophils across polycarbonate or nitrocellulose filters and that this response involved chemotaxis. The concentration of G-CSF required for neutrophil migration by Wang et al. was >10-100 U/ml (7-70 ng/ml). The discrepancy of G-CSF concentration for neutrophil migration compared with the present study might be due to the differences in neutrophil separation and solutions used for neutrophil suspension because human recombinant G-CSF induced neutrophil migration at 10-1,000 pg/ml in the present study. The concentrations of G-CSF in the A549 cell supernatant fluid in response to a variety of stimuli reached this chemotactic concentration.
The capacity of G-CSF to act as a chemoattractant for neutrophils may have in vivo relevance. G-CSF activity is produced by various cell types including stimulated lymphocytes, activated macrophages (4, 18), fibroblasts (2), and endothelial cells (12) exposed to mononuclear phagocyte products. Therefore, it is conceivable that G-CSF production, triggered in these cell types directly by exogenous materials (endotoxin or antigens) or indirectly via monokine release, might serve to rapidly recruit neutrophils from the blood compartment to local inflammatory sites.
In conclusion, A549 cells released G-CSF as NCA in response to IL-1,
TNF-, LPS, and BK. The expression of G-CSF mRNA was augmented in
response to each stimulus. These data suggest that ATII cells may
produce G-CSF as NCA and may participate in the regulation of
neutrophil extravasation and lung inflammation.
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FOOTNOTES |
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Address for reprint requests: S. Koyama, The First Dept. of Internal Medicine, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto 390, Japan.
Received 8 September 1997; accepted in final form 11 June 1998.
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)]; lane 2, 2 h of
incubation; lane 3, 4 h of incubation; lane 4, 8 h of incubation; lane 5, 12 h of incubation; lane
6, control (cont). G-CSF/beta actin, ratio of G-CSF to 