Human lung fibroblasts release chemokinetic
activity for monocytes constitutively
Sekiya
Koyama1,2,
Etsuro
Sato1,
Tsuyoshi
Masubuchi1,
Akemi
Takamizawa1,
Hiroshi
Nomura1,
Keishi
Kubo1,
Sonoko
Nagai2, and
Takateru
Izumi2
1 Shinshu University School of Medicine, First
Department of Internal Medicine, Matsumoto 390; and
2 Kyoto University Chest Disease Research
Institute, Kyoto 606-01, Japan
 |
ABSTRACT |
We determined whether
human lung fibroblasts (HLFs) might release mediators that are
responsible for monocyte chemokinetic activity (MCA) constitutively.
HLF supernatant fluids showed MCA in a time-dependent manner
(P < 0.001). Checkerboard analysis of 24- and 72-h
supernatant fluids showed that the activity was chemokinetic. Partial
characterization of 24- and 72-h supernatant fluids revealed that the
mediators released after 24 h were predominantly composed of
lipid-soluble activity, and MCA was blocked by lipoxygenase inhibitors.
The mediators released after 72 h were predominantly trypsin sensitive
and blocked by cycloheximide. Molecular-sieve column chromatography
identified four peaks of MCA. A polyclonal antibody to monocyte
chemoattractant protein-1 (MCP-1) inhibited MCA by 20% after 24 h and
by 40% after 72 h. Granulocyte-macrophage colony-stimulating factor
(GM-CSF) and transforming growth factor-
(TGF-
) antibodies
attenuated MCA released after 72 h by 30 and 10%, respectively. These
antibodies inhibited corresponding molecular-weight peaks separated by
molecular-sieve column. The concentrations of MCP-1, GM-CSF, and
TGF-
were 4,698 ± 242, 26.8 ± 3.8, and 550 ± 15 pg/ml,
respectively. A leukotriene B4
(LTB4)-receptor antagonist attenuated the total MCA and
the lowest molecular weight peak of MCA. The concentrations of
LTB4 were 153.4 ± 12.4 (24 h) and 212 ± 16.6 (72 h)
pg/ml. These findings suggest that HLFs may modulate the recruitment of
monocytes into the lung by releasing MCP-1, GM-CSF, TGF-
, and
LTB4 constitutively.
monocyte chemoattractant protein-1; granulocyte-macrophage colony-stimulating factor; transforming growth
factor-
; leukotriene B4
 |
INTRODUCTION |
THE NORMAL LUNG contains a resident population of
macrophages within intravascular, interstitial, and alveolar spaces.
These cells have a physiological role as part of the host defense of the lung (25, 42). Unlike other residential macrophages, they may be
less important as antigen-presenting cells but may retain an important
immune function (13, 14, 33). The macrophage may have an influence both
as an activating cell of the immune process and as a cell that can
terminate inflammatory responses. Although proposed mechanisms
responsible for persistent maintenance of these phagocytic cells within
the lung include chemotaxis (6, 18), local proliferation (1, 11), and
migration inhibition (42), macrophages predominantly originate from
peripheral blood monocytes that migrate into the lung (2, 18, 39, 41).
The fibroblast is the principal cell of most connective tissues and is
involved in constituting collagenous and noncollagenous components of
the extracellular matrix. This synthetic activity serves
an important structural function by providing a frame network for organ
integrity. In addition to this traditionally accepted function,
recent studies have demonstrated that fibroblasts not only serve to
maintain the connective tissue but are important participants in the
orchestration of acute and chronic inflammation. In this context,
fibroblasts released monocyte chemoattractant protein-1 (MCP-1),
granulocyte-macrophage colony-stimulating factor (GM-CSF), and
transforming growth factor-
(TGF-
) in response to interleukin-1
(IL-1), tumor necrosis factor-
(TNF-
), and platelet-derived
growth factor (PDGF), suggesting a contribution to certain disease
states (22, 23, 26, 36, 46). Therefore, the fibroblast, because of its
anatomic location, is in a pivotal position to participate in and to
direct bidirectional communications between interstitial and vascular
events.
Koyama and colleagues (20, 21) previously reported that
type II pneumocytes and bronchial epithelial cells release monocyte chemoattractant activity constitutively, suggesting participation of
these cells in monocyte recruitment as an origin of alveolar macrophages in the steady state. Although these epithelial cells may
play a role in monocyte migration from the interstitium to the alveolar
and bronchial spaces, the underlying mechanisms of monocyte migration
from the vascular compartment to the interstitium remains to be
elucidated. The role of human lung fibroblasts (HLFs) in monocyte
recruitment from the vascular compartment to the interstitium in the
steady state is uncertain.
The objective of the present study was to determine whether HLFs may
release mediators that are responsible for monocyte chemokinetic activity (MCA) constitutively. The results demonstrated that HLFs released MCA and that MCA involved GM-CSF, TGF-
, MCP-1, and
leukotriene B4 (LTB4). The present
investigation suggested that HLFs may contribute to the maintenance of
host defense and the lung immune environment by recruiting monocytes
into the lung interstitium.
 |
METHODS |
Preparation of HLFs.
We used fetal HLFs (diploid, passage 27), which were
established as a cell line and commercially available (American Type Culture Collection, Rockville, MD). HLFs were suspended at 1.0 × 106 cells/ml in F-12 medium supplemented with penicillin
(50 U/ml; GIBCO BRL, Grand Island, NY), streptomycin (50 µg/ml; GIBCO), Fungizone (2 µg/ml; GIBCO),
and 10% FCS (GIBCO). HLF suspensions (3 ml) were added to
a 30-mm-diameter tissue culture dish (Corning, Corning, NY) and
cultured at 37°C in a 5% CO2 atmosphere. After 4-6
days in culture, the cells had reached confluence, and then the culture
medium was replaced with 2 ml of medium supplemented as above and
incubated for 1 more day. On the following day, the culture was washed
two times; the culture medium was replaced with 1 ml of serum-free F-12
medium with penicillin, streptomycin, and Fungizone; and the culture
supernatant fluids were harvested at 12, 24, 48, 72, and 96 h and
frozen at
80°C until assayed for MCA. At least six separate HLF
culture supernatant fluids were harvested for each experiment.
Monocyte chemotaxis assay.
Blood was drawn from healthy adults into heparinized syringes by
venipuncture. Mononuclear cells were obtained for monocyte chemotaxis
by Ficoll-Hypaque density centrifugation (Histopaque-1077, Sigma) to
separate the red blood cells and neutrophils from the mononuclear
cells. The mononuclear cells were harvested at the interface, and the
red blood cells were lysed by suspending the cells in a solution
consisting of 0.1% KHCO3 and 0.83% NH4Cl. The
suspension was then centrifuged at 400 g for 10 min and washed three times in Hanks' balanced salt solution (Biofluids, Rockville, MD). The preparation routinely consisted of 30% large monocytes and
70% small lymphocytes as determined by morphology and
-naphthyl acetate esterase staining (Sigma), with >98% viability as assessed by trypan blue and erythrocin 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 5.0 × 106 cells/ml. This suspension was then used in the
chemotaxis assay.
The chemotaxis assay was performed in duplicate in 48-well
microchemotaxis chambers (NeuroProbe, Cabin John, MD) as described previously (8). The bottom wells of chambers were filled with 25 µl
of fluid containing the chemotactic stimulus. A polycarbonate filter
with a pore size of 5 µm (Nuclepore, Pleasanton, CA) was placed over
the bottom wells. The silicon gasket and top pieces of the chambers
were applied, and the entire assembly was preincubated at 37°C in
humidified air for 15 min before the top wells were filled with 50 µl
of the cell suspension. The chamber was incubated for 90 min in 5%
CO2 in humidified air at 37°C. After incubation, the
chamber was disassembled, and the filter was removed. The filter was
then fixed, stained with Diff-Quik (American Scientific Product, McGaw
Park, IL), and mounted on a glass slide. Mononuclear cells that
completely migrated through the membrane were counted in 10 random
high-power fields (HPFs; ×1,000). Chemotactic response was defined as
the mean number of migrated cells per HPF for each group. F-12 medium
supplemented as above but without FCS and incubated identically to the
HLF cultures was used to determine background monocyte migration.
Formyl-methionyl-leucyl-phenylalanine (10
7 M in
supplemented F-12 medium; Sigma) and normal human serum that was
activated by incubation with bacterial endotoxin (Escherichia coli lipopolysaccharide 0127:B8; DIFCO, Detroit MI) and diluted 10-fold with F-12 medium were used as positive controls (21).
To determine whether the migration of monocytes was because of movement
along a concentration gradient (chemotaxis) or stimulation of the
monocytes to randomly migrate (chemokinesis), a checkerboard analysis
was performed (47). To do this, various concentrations of HLF
supernatant fluid (1:1, 1:4, 1:16, 1:64, and 1:254) were placed with
mononuclear cells above the membrane and supernatant fluids with
similarly diluted concentrations were placed below the membrane. Thus
monocyte migrations were tested by a variety of concentration gradients
of supernatant fluid across the membrane.
To ensure that monocytes, but not lymphocytes, were the primary cells
that migrated, some membranes were stained with
-naphthyl acetate
esterase according to the manufacturer's directions (Sigma).
Partial characterization of MCA. Because MCA could be detected
in HLF culture supernatant fluid, a partial characterization of the
released activity was performed with supernatant fluid harvested at 24 and 72 h. Sensitivity to protease was tested by incubating the HLF
culture supernatant fluid with trypsin (final concentration 100 µg/ml; Sigma) for 30 min at 37°C followed by the addition of a 1.5 M excess of soybean trypsin inhibitor to terminate the proteolytic
activity before the chemotaxis assay. The lipid solubility of the
activity was evaluated by mixing the HLF supernatant fluid two times
with ethyl acetate, decanting the lipid phase after each extraction,
evaporating the ethyl acetate to dryness, and resuspending the
extracted material in the F-12 medium used for cell culture before the
chemotaxis assay. Heat sensitivity was determined by heating the
culture supernatant fluid at 98°C for 30 min.
To further examine the release of MCA, cycloheximide (10 µg/ml;
Sigma) was added to the supplemented F-12 medium without FCS to inhibit
protein synthesis (9), and nordihydroguaiaretic acid (NDGA; 100 µM;
Sigma), diethylcarbamazine (DEC; 1 mM; Sigma), and AA-861 (100 µM;
Takeda Pharmaceutical, Tokyo, Japan) were added to inhibit
lipoxygenase-derived arachidonic acid metabolite release (16). The HLF
culture was incubated with cycloheximide and the lipoxygenase
inhibitors for 24 and 72 h and harvested, and the supernatant fluid was
assayed for MCA.
To determine the approximate molecular weight of the released MCA,
molecular-sieve column chromatography was performed with Sephadex G-100
(75 × 1.75 cm; Pharmacia, Piscataway, NJ) at a flow rate of 12 ml/h. The HLF cell supernatant fluid was eluted with phosphate-buffered
saline, and every fraction after the void volume was evaluated in
duplicate for monocyte chemotactic activity.
Effects of LTB4- and platelet-activating
factor-receptor antagonists.
LTB4-receptor (ONO-4057, Ono Pharmaceutical, Tokyo, Japan)
and platelet-activating factor (PAF)-receptor (TCV-309, Takeda Pharmaceutical) antagonists at a concentration of 10
5 M
were used to evaluate the responsible chemotactic activity in the
supernatant and column chromatography-separated lowest molecular weight
peak. These receptor antagonists completely blocked the chemotactic
responses of 10
7 M LTB4 and PAF but did not
have any influence on the chemotactic response of monocytes to
endotoxin-activated serum (data not shown).
Measurement of LTB4 and PAF by radioimmunoassay.
The measurement of LTB4 and PAF in HLF supernatant
fluid harvested at 24 and 72 h was performed by RIA, as previously
described (21), with anti-LTB4 serum,
[5,6,8,9,11,12,14,15-3H]- LTB4, and
synthetic LTB4, which were purchased from Amersham (Arlington Heights, IL). Briefly, ethanol samples were centrifuged at 5,500 g at 0°C. The supernatants were evaporated under
N2 gas at 37°C. To each sample, 10 ml of distilled water
were added. These samples were acidified to pH 4.0 with 0.1 N
HCl and were applied to Sep-Pak C18 columns (Waters
Associates, Milford, MA); the columns were washed with 10 ml of
distilled water and 20 ml of petroleum ether and then were eluted with
15 ml of methanol. These eluates were dried with N2 gas at
37°C and redissolved in 20 µl of methanol and 180 µl of RIA
buffer [50 mM Tris · HCl buffer containing 0.1% (wt/vol)
gelatin, pH 8.6]. [3H]LTB4 was diluted in
RIA buffer, and 100-µl aliquots containing 4,000 disintegrations/min
were mixed with 100 µl of standard or sample in siliconized tubes.
Anti-LTB4 serum diluted in RIA buffer (100 µl) was added
to give a total incubation volume of 400 µl, and the mixture was
incubated at 4°C for 18 h. Free LTB4 was adsorbed onto
dextran-coated charcoal. The supernatant containing the antibody-bound LTB4 was decanted into a scintillation counter after
centrifugation at 2,000 g for 15 min. Scintillation fluid was
added, and the radioactivity was counted by a scintillation counter
(Tricarb-3255, Packard, Downers Grove, IL) for 4 min.
PAF concentration in the supernatant fluid was measured by a
scintillation-proximity assay system. This system combined the use of a
high specific activity tritiated-PAF tracer with an antibody specific
for PAF and a PAF standard, similar to the methods used for the
measurement of LTB4.
Effects of regulated on activation normal T cells expressed and
secreted, GM-CSF, TGF-
, and MCP-1 polyclonal antibodies
on the released chemotactic activity.
The neutralizing antibodies to regulated on activation normal T cells
expressed and secreted (RANTES), GM-CSF, TGF-
, and MCP-1 (purchased
from Genzyme, Cambridge, MA) were added to the supernatant fluids
harvested at 24 and 72 h at the suggested concentrations to inhibit
these cytokines and incubated for 30 min at 37°C. Then these samples
were used for chemotactic assay. These antibodies did not influence the
chemotactic response to endotoxin-activated serum (data not shown).
Measurement of RANTES, GM-CSF, TGF-
, and MCP-1
concentrations in the supernatant fluid.
The concentrations of RANTES, GM-CSF, TGF-
, and MCP-1
in the
supernatant fluids harvested at 24 and 72 h were measured by ELISA
according to the manufacturer's directions. GM-CSF and RANTES kits
were purchased from Amersham (Amersham, UK), and the minimum concentration detected by these methods was 2.00 pg/ml for GM-CSF and
15.6 pg/ml for RANTES. MCP-1 and TGF-
kits were purchased from R&D
Systems (Minneapolis, MN), and the minimum concentration for MCP-1 and
TGF-
was 31.3 pg/ml and 0.31 ng/ml, respectively.
Statistical analysis.
Differences in the chemotactic activity were tested for significance
with a two-tailed Student's paired t-test. A P value < 0.05 was considered significant. Data are expressed as means ± SD.
 |
RESULTS |
Release of MCA from HLFs.
Six different HLF culture supernatant fluids were evaluated. Each
culture contained MCA that was significantly increased compared with
the supplemented F-12 medium alone (P < 0.001; Fig.
1). MCA was released into the culture
medium in a time-dependent manner, reaching a plateau at 72 h (Fig.
2). Checkerboard analysis revealed that
supernatant fluids harvested after both 24 and 72 h of culture induced
increasing migration with increasing concentrations of supernatant
fluid in the absence of a gradient across the membrane (data not
shown). Thus MCA in both the 24- and 72-h supernatant fluids was
consistent with chemokinetic activity rather than chemotactic activity.

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Fig. 1.
Demonstration that human lung fibroblasts (HLFs) release monocyte
chemokinetic activity (MCA). CONT, supplemented F-12 medium alone
(control); FMLP, formyl-methionyl-leucyl-phenylalanine. Dots connected
by a line are matching monocyte chemotaxis experiments from 6 different
HLF culture supernatant fluids.
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Fig. 2.
Time course of MCA release from HLF cultures. Values are means ± SE;
n = 7 experiments. MCA increased in a time-dependent manner and
reached a plateau at 72 h. * P < 0.05 compared with F-12
medium (0 h).
|
|
Confirmation that monocytes were the primary cells attracted was
provided by the following three lines of evidence: 1) >90% of the migrated cells appeared to be monocytes by light microscopy, 2) >90% of the migrating cells were esterase positive, and
3) lymphocytes prepared by incubating mononuclear cells
recovered by Ficoll-Hypaque density centrifugation on plastic dishes
(Costar no. 3424, Costar, Cambridge, MA) for 60 min at 37°C and
tested for chemotactic activity as above resulted in 0-20% of the
activity of the mononuclear cells not depleted of monocytes.
Partial characterization of MCA.
The MCA was heterogeneous in its character. The MCA released at 24 h
was predominantly sensitive to heat, extractable into ethyl acetate,
and partially digested by trypsin (Fig.
3A). In contrast, MCA released at
72 h was predominantly sensitive to heat, digested by trypsin, and
partially extractable into ethyl acetate (Fig. 3B).
Incubation of HLFs with cycloheximide inhibited the release of MCA more
potently at 72 h than at 24 h. The nonspecific lipoxygenase inhibitors
NDGA and DEC and the 5-lipoxygenase inhibitor AA-861 inhibited the
release of MCA at 24 h rather than at 72 h (Fig.
4). Cycloheximide, NDGA, DEC, and AA-861
themselves did not inhibit monocyte migration in response to HLF
supernatant fluid harvested after 72 h of incubation (data not shown).
Thus the effects of these agents were due to the inhibition of the release of MCA.

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Fig. 3.
Partial characterization of MCA release from HLFs. A:
supernatant fluid harvested at 24 h of incubation. B:
supernatant fluid harvested at 72 h of incubation. F-12, medium alone.
Values are means ± SE; n = 6 experiments. * P < 0.01 compared with crude supernatant fluid.
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Fig. 4.
Effects of incubation of HLFs with cycloheximide, nordihydroguaiaretic
acid (NDGA), diethylcarbamazine (DEC), and AA-861 on release of MCA.
A: supernatant fluid harvested at 24 h of incubation.
B: supernatant fluid harvested at 72 h of incubation. Values
are means ± SE; n = 7 experiments. * P < 0.01 compared with crude supernatant fluid.
|
|
Molecular-sieve column chromatography revealed that MCA was
heterogeneous in size (Fig. 5). At least
four peaks of MCA were separated by column chromatography, with the
estimated molecular weight before that of BSA and slightly before that
of cytochrome c (mol wt 12,384) and with an additional peak
that eluted near quinacrine (mol wt 565). At 72 h of incubation, the
second and third molecular-weight peaks became prominent (Fig.
5B).

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Fig. 5.
Molecular-sieve column chromatography with Sephadex G-100 of HLF
supernatant fluid harvested after 24 (A) and 72 h
(B) of incubation. Arrows and nos. on top, position
and molecular weight, respectively, of markers for BSA (66,000),
cytochrome c (12,400), and quinacrine (450). This is a
representative profile of 4 experiments.
|
|
Effects of LTB4- and PAF-receptor antagonists on MCA and
concentrations of LTB4 and PAF in supernatant fluid.
LTB4 and PAF were potent chemotactic activators for
monocytes; thus the effects of LTB4- and PAF-receptor
antagonists were evaluated. LTB4-receptor antagonists
inhibited the total MCA to 60% released after 24 h and to 40%
released after 72 h in the supernatant fluid (Fig.
6) and also inhibited the column
chromatography-separated lowest molecular weight peak of MCA released
after 72 h to 60% (34.5 ± 2.4 vs 16.3 ± 3.2 cells/HPF;
P < 0.001). The release of LTB4 in the
supernatant fluid after 24 and 72 h of incubation was 154.3 ± 12.4 and 212.0 ± 16.6 pg/ml, respectively. These concentrations of
synthetic LTB4 induced monocyte migration
(10
9 M, 35.4 ± 5.6 cells/HPF; 10
10 M,
27.3 ± 4.3 cells/HPF). In contrast, the PAF-receptor antagonist did
not block the chemotactic response, and PAF was not detected in both
the 24- and 72-h supernatant fluids.

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Fig. 6.
Effects of leukotriene B4 (LTB4)- and
platelet-activating factor (PAF)-receptor antagonists on supernatant
fluid harvested at 24 (A) and 72 h (B) of
incubation. Values are means ± SE; n = 6 experiments.
* P < 0.01 compared with untreated supernatant fluid.
|
|
Effects of GM-CSF, RANTES, TGF-
, and MCP-1
antibodies on MCA.
The effects of blocking polyclonal antibodies to GM-CSF, RANTES,
TGF-
, and MCP-1 on MCA released after 24 h revealed that the MCP-1
antibody significantly inhibited MCA in the supernatant fluid (Fig.
7A). The polyclonal antibodies to
MCP-1, GM-CSF, and TGF-
attenuated MCA in 72-h supernatant fluid up
to 40, 30, and 10%, respectively (Fig. 7B). In contrast,
RANTES antibodies did not show any effects for both the 24- and 72-h
supernatant fluids. Furthermore, the corresponding molecular-weight
peaks of MCA after 72 h, as separated by a molecular-sieve column, were
inhibited by MCP-1, GM-CSF, and TGF-
polyclonal antibodies up to
60-70%, respectively.

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Fig. 7.
Effects of monocyte chemoattractant protein-1 (MCP-1), transforming
growth factor- (TGF- ), granulocyte-macrophage colony-stimulating
factor (GM-CSF), and regulated on activation normal T cells expressed
and secreted (RANTES) polyclonal antibodies on released MCA released
after 24 (A) and 72 h (B). Values are means ± SE; n = 6 experiments. * P < 0.01 compared with untreated
supernatant fluid.
|
|
Concentrations of GM-CSF, RANTES, TGF-
, and MCP-1 in
the supernatant fluid.
The concentrations of MCP-1, GM-CSF, and TGF-
evaluated by ELISA in
the supernatant fluid after 24 and 72 h of incubation were 1,863 ± 214, 7.3 ± 2.1, and <310 pg/ml (not detected) (24 h) and 4,698 ± 242, 26.8 ± 3.8, and 550 ± 15 pg/ml (72 h), respectively. There was
no detectable amount of RANTES in the supernatant fluid harvested at
both 24 and 72 h.
 |
DISCUSSION |
The present study demonstrates that HLFs can release MCA. The partial
characterization of MCA revealed that the activity was composed of a
low- molecular-weight lipid-soluble activity and high-molecular-weight
peptides after both 24 and 72 h in culture. The MCA was partly
inhibited by MCP-1, GM-CSF, and TGF-
antibodies and an
LTB4-receptor antagonist. The concentration of
LTB4 was almost 10
9 M. MCP-1, GM-CSF, and
TGF-
were detected by ELISA of the supernatant fluid. These
concentrations were high enough for monocyte migration. These data
suggest that lung fibroblasts may modulate their local environment by
releasing MCA.
Previous studies addressing monocyte influx into the alveolar space
have yielded conflicting results. Collins and Auclair (5), studying
unstimulated parabiotic rats, found no evidence for radiolabeled
monocyte influx into the lungs of the recipient rat after
injecting the donor rat monocytes with tritiated thymidine. Other
studies have found no changes in alveolar macrophage cell number in
animals with radiation-induced monocytopenia (32), also suggesting that
the normal alveolar macrophage population is at least partially
independent of circulating blood monocytes. Similar observations have
been made in humans with severe monocytopenia (37, 38). In contrast,
Blusse Van Oud Albas and Van Furth (2), employing multiple injections
of tritiated thymidine into normal mice, observed simultaneous
radiolabeling in the blood monocyte and alveolar macrophage
populations. This coincident radiolabeling was interpreted to mean that
the labeled alveolar macrophages were directly derived from the blood
monocytes. Bowden and Adamson (3) radiolabeled normal mice with
multiple injections of tritiated thymidine and observed heavily labeled
cells among the alveolar macrophages. Furthermore, support for a bone
marrow origin of alveolar macrophages has come from cell marker studies in mouse radiation chimeras and from studies in humans after bone marrow and heart-lung transplantation (9, 28, 39). Our study
demonstrated that MCA released from HLFs facilitated the monocyte
recruitment into the lung, supporting the concept that alveolar
macrophages are at least partly derived from circulating blood
monocytes.
Macrophages may have an influence as an activating cell of the immune
responses by weakly presenting antigens to lymphocytes during the
development of specific immunity (31, 40) and releasing IL-1 (13, 14),
a mediator that is crucial for the initiation of lymphocyte activation.
Previous studies (22, 35, 36, 46) have shown that dermal and synovial
fibroblasts can release soluble chemotactic factors that direct the
migration of neutrophils and monocytes into the alveolar space in
response to TNF-
, IL-1, and PDGF, and the present study demonstrated
that HLFs also release MCA constitutively. In this context, HLFs may
modulate their local immunologic environment by releasing chemotactic
activity for both neutrophils and monocytes.
The lung population of macrophages continually exits from the
interstitium to the airway (10). This perpetual movement of phagocytic
cells from the alveolar lesions cleanses the lung and aids in the
maintenance of lung sterility (4, 12). Forces that attract macrophages
to the interstitium and direct macrophages from the interstitium to the
distal alveolar surface areas and further to the central airways are
poorly understood. Type II pneumocytes and bronchial epithelial cells
release chemoattractant activity for alveolar macrophages (20, 21),
which may serve as one of the cellular sources of lung lining material
and may attract alveolar macrophages toward the central airways. In
contrast, HLFs may release MCA and may maintain the population of
macrophages in the lung interstitium.
The identification of MCA released from HLFs is not complete. However,
the trypsin sensitivity of MCA along with the inhibition of the release
by cycloheximide treatment suggests that the activity is at least
partly dependent on protein synthesis (10). In contrast, extractability
of MCA into ethyl acetate along with the inhibition of release by NDGA,
DEC, and AA-861 suggests that the activity is composed of lipid. The
released activity was attenuated by antibodies to MCP-1, GM-CSF, and
TGF-
. The lipid-extractable activity was inhibited by the
LTB4-receptor antagonist. The concentrations of MCP-1,
GM-CSF, and TGF-
in the supernatant fluid reached the concentrations
as monocyte chemotactic activity (34, 43, 44). Thus HLFs at least
partly released LTB4, MCP-1, GM-CSF, and TGF-
as
responsible MCA.
Production of chemotactic factors for monocytes by cells other than
HLFs may also be important in regulating monocyte recruitment to the
lung. Numerous cells that produce monocyte chemotactic activity are
present in the lung, and a wide variety of molecules including
fibronectin fragments (27), elastin fragments (17), collagen and
collagen fragments (29), complement-derived chemoattractants (20), and
surfactant proteins (15, 45) has been identified that may serve as
chemoattractants in certain lung disorders. One cell that can release
monocyte chemotactic activity is the alveolar macrophage, which can
release PDGF (7, 24), GM-CSF (34), TGF-
(43), and lipoxygenase
products (25). Other cells associated with lung tissues, such as
bronchial epithelial cells, type II pneumocytes, and lymphocytes, may
also release MCA, including TGF-
, GM-CSF, MCP-1, RANTES and
lipoxygenase products (19-21, 30). Further studies clarifying the
role of HLFs and purifying and identifying MCA produced by HLFs should
make the relevance of these known chemoattractants clear. Furthermore, the actual migratory responses of monocytes in a complex biological fluid such as lung epithelial lining fluid likely involves complex interactions among factors. It is reasonable to suggest that
interactions among chemokinetic, chemotactic, and migration inhibitory
factors may determine monocyte movement in vivo (42).
The present investigation demonstrates that HLFs can release MCA
constitutively. Although these data are derived from HLFs in in vitro
conditions, HLFs may contribute to the maintenance of host defense by
modulating the population of lung macrophages.
 |
FOOTNOTES |
Address for reprint requests: S. Koyama, First Department of
Internal Medicine, Shinshu Univ. School of Medicine, 3-1-1 Asahi,
Matsumoto 390, Japan.
Received 21 July 1997; accepted in final form 27 April 1998.
 |
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