Vol. 84, Issue 5, 1528-1534, May 1998
Acetylcholine and substance P stimulate bronchial epithelial
cells to release eosinophil chemotactic activity
Sekiya
Koyama1,2,
Etsuro
Sato1,
Hiroshi
Nomura1,
Keishi
Kubo1,
Sonoko
Nagai2, and
Takateru
Izumi2
1 The First Department of Internal Medicine,
Shinshu University School of Medicine, Matsumoto 390; and
2 Kyoto University Chest Disease Research
Institute, Kyoto 606-01, Japan
 |
ABSTRACT |
We
investigated a role of neuroregulation in the release of eosinophil
chemotactic activity (ECA) from bovine bronchial epithelial cells
(BBEC). BBEC were stimulated with acetylcholine (ACh) and substance P
(SP), and the supernatant fluids were tested for ECA by a blind-well
chemotactic chamber technique. BBEC released ECA in response to ACh and
SP in a dose- and time-dependent manner. Checkerboard analysis showed
that ECA in regard to ACh and SP was chemotactic rather than
chemokinetic. Partial characterization revealed that ECA involved both
lipids and peptides. The release of ECA in response to ACh and SP was
inhibited by nonspecific and 5-specific lipoxygenase inhibitors and by
cycloheximide (P < 0.01). Molecular-sieve column
chromatography revealed that these mediators induced three molecular
mass peaks (near 25 kDa, 9 kDa, and 400 Da, respectively). The lowest
peak, which represented the predominant activity, was blocked by
leukotriene B4-receptor antagonist (P < 0.01) but not by platelet-activating factor-receptor antagonist. The release
of leukotriene B4 in the supernatant fluids was increased in response to ACh and SP stimulation (P < 0.01).
Platelet-activating factor was not detected. These results raise the
possibility of a role of neuroregulation for the elaboration of ECA in
the airway.
bronchial epithelial cell; eosinophil chemotaxis; leukotriene
B4
| |
INTRODUCTION |
AIRWAY EOSINOPHILIC INFLAMMATION is one of the most
specific changes in the pathology of asthma (10) and a major marker of
the inflammatory activity in the disease (31). An increased number of
eosinophils is usually found in the sputum (13), in the bronchoalveolar
lavage fluid (BALF) (8, 37), in the airway epithelia and submucosa (5,
31), and, frequently, in the blood (21) of asthmatic patients. Although
the lymphocytes and the eosinophils are the predominant infiltrating
cells in airway mucosa of asthmatic subjects (10), the eosinophil is
believed to be a primary cell responsible for the development of many
features of asthma, including damage and desquamation of the
respiratory epithelia (7), airway hyperresponsiveness (37), and
allergen-induced late asthmatic reaction (8). Furthermore, the degree
of infiltration of the bronchial wall by eosinophils is related to the
clinical severity of asthma (6).
The extent of eosinophil accumulation at a specific site in the
organism is regulated by several mechanisms. One requirement is the
availability of a sufficient number of cells in the blood pool.
Although the mechanisms that govern the egress of eosinophils from the
bone marrow are not fully understood, T-lymphocyte-dependent and
-independent mechanisms have been proposed (4). The second major
requirement is the production of some factors at the specific site,
which signal the eosinophil to leave the bloodstream. The administration of antibody to one of the adhesion molecules,
intercellular adhesion molecule-1, inhibited the eosinophil
accumulation and the development of hyperreactivity, suggesting a role
of the eosinophil-endothelial cell interaction (38). However, it is
assumed that these signals involve specific eosinophil chemotactic
factors (16).
Because the actions of substance P (SP) include vasodilation, vascular
leakage, airway secretion, and contraction of smooth muscles (2, 3) and
because acetylcholine (ACh) regulates airway tone and secretion, SP and
ACh are thought to be potent factors in asthma (3). Bovine bronchial
epithelial cells (BBEC) participate in the inflammatory response in the
airway by releasing chemotactic activity for neutrophils and monocytes
(26-29, 36). However, the role of neuroregulation of the airway
cells for the elaboration of eosinophils is unknown. In the present
study, we demonstrated that BBEC released eosinophil chemotactic
activity (ECA) in response to ACh and SP. These results may suggest the possibility of a role of neuroregulation for the elaboration of ECA, in
addition to the known neural involvement in airway pathophysiology of
asthma.
| |
METHODS |
Culture and identification of BBEC.
BBEC were isolated and cultured by a modification of the methods of Wu
and colleagues (39). Briefly, bovine lungs were obtained from freshly
killed cows. The alveolar and interstitial structures were removed by
teasing, and the bronchi were sectioned into 4- to 7-cm-long pieces.
The sectioned bronchi were then incubated overnight at 4°C in
Eagle's minimum essential medium (GIBCO, Grand Island, NY) with 0.1%
bacterial protease (Streptomyces griseus, type XIV; Sigma
Chemical, St. Louis, MO). The BBEC were recovered by washing the
epithelial surface of the bronchi. The recovered cells were suspended
at 2.0 × 106 cells/ml in medium 199 (GIBCO) supplemented
as previously described (28). Then 1.5 ml of BBEC suspension were added
to a 35-mm-diameter tissue-culture dish (Corning, Corning, NY) and
cultured at 37°C in 5% CO2 atmosphere. The
tissue-culture dish was not coated with any types of substratum of
extracellular matrix protein. With the use of these techniques, the
cultured cells were identified as epithelial cells by staining with
anti-keratin antibody (ICN Immunological, Lisle, IL), and these cells
were histologically and functionally competent as previously reported
(28, 29). BBEC monolayers cultured as above formed dome by
phase-contract microscope, and tight junctions were assessed by
electron microscope and blocked albumin permeability. Transepithelial
resistance was 412-487 
· cm2 (457 ±
32 · cm2, n = 6 experiments)
cultured on Transwell plates.
Exposure of BBEC to ACh and SP.
Medium was removed from cells by washing them twice with serum-free
medium, and BBEC were incubated with medium supplemented without fetal
calf serum (FCS; GIBCO), both in the presence and absence of ACh and SP
(0, 0.1, 1.0, 10, 100, and 1,000 µM, respectively) and cultured at
37°C in a humidified 5% CO2 atmosphere for 4, 6, 12, 24, 48, and 72 h. These agents did not cause BBEC injury (no
deformity of cell shape, no detachment from tissue-culture dish, and
>95% of cells viable by trypan blue exclusion) after 72-h incubation
at the maximal doses.
Because BBEC made domes, formed tight junctions, and polarized (26,
27), we cultured BBEC on Transwell plates, which had 24 wells and were
separated by polycarbonate filters that had a diameter of 6.5 mm and a
pore size of 0.3 µm. After cells reached confluence and BBEC
monolayers had negligible BSA permeability compared with filter alone,
BBEC were stimulated from the basolateral side with 100 µM of ACh and
SP for 48 h. The upper and lower chambers were filled with 500 µl of
serum-free medium to avoid pressure gradient. The supernatant fluids in
the upper and lower chambers were harvested after 48 h for chemotaxis
assay.
The culture supernatant fluids were harvested and frozen at 
80°C
until assayed. At least five separate BBEC supernatant fluids were
harvested from cultures obtained from different animals for each
experimental condition.
Measurement of ECA.
Eosinophil isolation was performed by a minor modification of the
method by Hansel and co-workers (18), who used a magnetic cell-separation system. The purity of eosinophils as counted by Randolph's stain was >94%, and the viability was >98%. Isolated eosinophils were washed twice with PIPES buffer containing 1% FCS. The eosinophils were suspended in Gey's balanced
salt solution containing 2% BSA at pH 7.2 to give a final
concentration of 3.0 × 106 cells/ml. These suspensions
were used in the chemotaxis assay.
The chemotaxis assay was performed in 48-well microchemotaxis chamber
(Neuroprobe, Cabin John, MD), as has been described (19). Each sample
was tested in duplicate. A polycarbonate filter (Nucleopore,
Pleasanton, CA) with a pore size of 5 µm was placed, and the chamber
was incubated in humidified 5% CO2 at 37°C for 120 min.
After the incubation, the chamber was disassembled, and the filter was
fixed, stained with Diff-Quik (American Scientific Products, McGraw
Park, IL), and mounted on a glass slide. Cells that completely migrated
through the filter were counted in five random high-power fields (HPF:
×1,000) from each duplicate well. Chemotactic response was defined as
the mean number of migrated cells per HPF. Medium 199 without FCS was
incubated identically with BBEC, and the supernatant fluids harvested
were used to determine background eosinophil migration. Leukotriene
B4 (LTB4; at 10
7 M in
supplemented medium 199; Sigma Chemical) and platelet-activating factor
(PAF; at 107 M in supplemented medium 199) were used as
positive controls for eosinophils.
To determine whether the migration was due to a movement along a
concentration gradient (chemotaxis) or to a stimulation to randomly
migrate (chemokinesis), a checkerboard analysis (40) was performed with
BBEC supernatant fluid stimulated by using 100 µM of ACh and SP for
72 h. To do this, various concentrations of BBEC supernatant fluids
(1:27, 1:9, 1:3, 1:1) were placed above and below the membrane.
Partial characterization of the released ECA.
Because ECA was detected in BBEC culture supernatant fluids, partial
characterization was performed by utilizing supernatant fluids
harvested at 72 h in response to ACh and SP. Sensitivity to protease
was tested by incubating BBEC culture supernatant fluids with trypsin
(final concentration 100 µg/ml; Sigma Chemical) 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 chemotactic
assessment. The lipid solubility was evaluated by mixing BBEC culture
supernatant fluids twice with ethyl acetate, decanting the lipid phase
after each extraction, evaporating the ethyl acetate to dryness, and
resuspending the extracted material in the supplemented medium 199. Heat sensitivity was determined by maintaining a temperature of 98°C
for 15 min.
Effects of metabolic determinants on ECA release.
BBEC are capable of releasing arachidonic acid metabolites that may
account for the released ECA. Therefore, BBEC were pretreated by
nonspecific and 5-specific lipoxygenase inhibitors:
nordihydroguaiaretic acid (NDGA; 100 µM, Sigma Chemical),
diethylcarbamazine (DEC; 1 mM, Sigma Chemical), and AA-861 (100 µM,
Takeda Pharmaceutical, Tokyo, Japan) for 30 min before the addition of
stimuli, and their effects on the release of ECA were examined. To
evaluate the role of protein synthesis in the release of ECA, BBEC were
pretreated with cycloheximide (10 µg/ml, Sigma Chemical). At these
concentrations, NDGA, DEC, and AA-861 inhibited the release of
lipoxygenase metabolites from BBEC in response to lipopolysaccharide
(26, 27). These inhibitors did not cause cytotoxicity to BBEC after
72-h incubation.
Partial purification of ECA by column chromatography.
To determine the approximate molecular mass of the
chemotactic activity released from BBEC in response to ACh and SP,
molecular-sieve column chromatography was performed by using Sephadex
G-75 (75 × 1.25 cm, Pharmacia, Piscataway, NJ). At a flow rate of 6 ml/h, the BBEC culture supernatant fluid was eluted with PBS, and every other fraction after the void volume was evaluated for ECA in duplicate.
To evaluate the responsible chemotactic activity in the lowest
molecular mass peak separated by column chromatography,
LTB4-receptor antagonist (ONO-4057, Ono Pharmaceutical,
Tokyo, Japan) and PAF-receptor antagonist (TCV-309, Takeda
Pharmaceutical) were used at the concentration of 105
(25, 35).
Measurement of LTB4 and PAF in the supernatant fluid.
The measurement of LTB4 was performed by radioimmunoassay
(RIA), according to manufacturer's directions (1), by using
anti-LTB4 serum [5, 6, 8, 9, 11, 12, 14, 15-3H(N)]LTB4 and synthetic LTB4,
which were purchased from Amersham (Arlington Heights, IL). Briefly,
ethanol samples were centrifuged at 5,500 g at 0°C. Then, the
supernatant fluids were evaporated under N2 gas at 37°C.
To each sample, 10 ml distilled water were added. These samples were
acidified to pH 4.0 with 0.1 M HCl and applied to Sep-Pak
C18 columns (Waters Associates, Milford, MA). The columns
were washed twice with a mixture of 10 ml distilled water and 20 ml
petroleum ether, then eluted with 15 ml ethanol. These eluates were
dried with N2 gas at 37°C, then redissolved in a
combination of 20 µl methanol and 180 µl 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 dpm were mixed with 100 µl
of standards or samples in disposable siliconized tubes.
Anti-LTB4 serum diluted in 100 µl of RIA buffer were added to give a total incubation volume of 0.4 ml.
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 scintillation
counter after centrifugation for 15 min at 2,000 g.
Scintillation fluid (Aquazol 2; DuPont NEN, Boston, MA) was added, and
radioactivity was counted by scintillation counter (Tricarb-3255,
Tackard, IL) for 4 min.
PAF concentration in the supernatant fluids was evaluated by the
scintillation-proximity assay system. This system combined the use of a
high-specific-activity tritiated PAF tracer, which included an antibody
specific for PAF and a PAF standard similar to the methods of
measurement of LTB4 (30).
Statistics.
In experiments that were multiphased, significant differences between
groups were tested by using one-way analysis of variance. Duncan's
multiple-range test was applied to data at specific time and dose
points. In experiments in which a single measurement was made, the
difference between groups was tested for significance by using
Student's paired t-test. In all cases, a P value
<0.05 was considered significant. Data in Figs. 1-7 and Tables 1
and 2 are expressed as means ± SE.
| |
RESULTS |
Release of ECA from BBEC.
BBEC released ECA in response to ACh and SP in a dose-dependent fashion
(Fig. 1, A and B). The
lowest dose that stimulated BBEC in response to ACh and SP was 0.1 µM. An increase in concentrations of ACh and SP progressively
augmented the release of ECA, culminating at 100 µM. The release of
ECA began 12 h after exposure to ACh and SP (Fig.
2, A and B), and the
released activity reached a plateau at 48 h (Fig. 2, A and
B). The released ECA harvested from BBEC on Transwell plates
from both chambers did not show significant difference [upper chamber
22.5 ± 3.2 (ACh), 20.7 ± 4.2 (SP) vs. lower chamber
18.4 ± 4.9 (ACh), 22.8 ± 4.6 (SP) cells/HPF, n = 6,
P > 0.05].
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Fig. 1.
Dose-dependent release of eosinophil chemotactic activity in response
to acetylcholine (ACh; A) and substance P (SP; B)
from bovine bronchial epithelial cell monolayer (n = 6
experiments). Values are means ± SE. * P < 0.05 compared
with supernatant fluids without stimuli.
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Fig. 2.
Time-related release of eosinophil chemotactic activity in response to
100 µM of ACh (A) and SP (B) from bovine
bronchial epithelial cell monolayer (n = 6 experiments).
Values are means ± SE. * P < 0.05 compared with
supernatant fluids without incubation.
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|
The chemotactic activities in response to LTB4 and PAF were
58.4 ± 8.7 and 68.3 ± 3.2 eosinophils/HPF, respectively. ACh and SP
by themselves did not show any chemotactic activity for eosinophils in
the culture medium without BBEC and when incubated identically (data
not shown).
Checkerboard analysis revealed that BBEC supernatant fluids stimulated
by ACh and SP induced eosinophil migration in the presence of
concentration gradient across the membrane and a smaller increase in
the absence of gradient (Tables 1 and
2). Thus the
migration in response to ACh- and SP-stimulated BBEC supernatant fluids was predominantly chemotactic, rather than chemokinetic.
Partial characterization of the released ECA.
The released chemotactic activity obtained from BBEC supernatant fluids
incubated with 100 µM of ACh and SP for 72 h was partially sensitive to heat and lipid extraction (Fig.
3, A and B). Trypsin digestion and ethyl acetate extraction enhanced the ECA as compared with the crude sample.
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Fig. 3.
Partial characterization of released eosinophil chemotactic activity in
response to ACh (A) and to SP (B) from bovine
bronchial epithelial cell monolayer (n = 6 experiments). EA
extracted, eosinophil chemotactic activity that was extracted into
ethyl acetate, dried, and resuspended in M-199 medium. EA extractant,
remaining eosinophil chemotactic activity in supernatant fluids after
ethyl acetate extraction. Values are means ± SE. * P < 0.01 compared with crude sample.
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Effects of metabolic inhibitors on the release of ECA.
BBEC incubated with 100 µM of ACh and SP in the presence of the
lipoxygenase inhibitors NDGA, DEC, and AA-861 showed a significant decrease in the release of ECA (Fig. 4,
A and B) into the supernatants. NDGA, DEC, and AA-861
did not have any effects on LTB4- and PAF-induced eosinophil chemotaxis (data not shown). Cycloheximide partially inhibited the release of ECA in response to ACh and SP (Fig.
4, A and B).
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Fig. 4.
Effects of nordihydroguaiaretic acid (NDGA), diethylcarbamazine (DEC),
AA-861, and cycloheximide on release of eosinophil chemotactic activity
in response to ACh (A) and to SP (B) from bovine
bronchial epithelial cell monolayer (n = 6 experiments).
Values are means ± SE. * P < 0.01 compared with stimulus
alone.
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Partial purification of ECA.
Molecular-sieve column chromatography with the use of Sephadex G-75
revealed that the released ECA was heterogeneous in size (Fig.
5, A and B). At least
three peaks of activity were identified by column chromatography, with
the estimated molecular mass after BSA (66.2 kDa), after
cytochrome-c (12.3 kDa), and an additional peak that eluted
near quinacrine (450 Da), respectively. The lowest molecular mass peak
near quinacrine represented the majority of the activity.
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Fig. 5.
Molecular-sieve column chromatographic findings of released eosinophil
chemotactic activity in response to ACh (A) and SP
(B) from bovine bronchial epithelial cell monolayer. Data are
a representative profile of 4 experiments.
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Effects of LTB4 and PAF-receptor antagonists on the
released ECA.
The activity of the lowest molecular mass ECA separated by G-75 column
chromatography was partially inhibited (>50%) by the addition of
LTB4-receptor antagonist ONO-4057 (Fig.
6, A and B). The
PAF-receptor antagonist, TCV-309, did not inhibit this lowest molecular
mass ECA. These receptor antagonists at the concentration of
105 M completely inhibited the eosinophil migration in
response to LTB4 and PAF at a concentration of
107 M (15.6 ± 2.4 and 13.4 ± 3.1
eosinophils/HPF, respectively) but showed no inhibitory
effects on eosinophil chemotaxis induced by activated serum (data not
shown).
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Fig. 6.
Effects of leukotriene B4 (LTB4) receptor
(ONO-4057; anti-LTB4) and platelet-activating factor (PAF)
(TCV-309; anti-PAF)-receptor antagonists on column
chromatography-separated lowest molecular mass eosinophil chemotactic
activity in response to ACh (A) and SP (B)
(n = 4 experiments). Values are means ± SE.
* P < 0.01 compared with crude
fraction.
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Concentrations of LTB4 and PAF in the supernatant
fluids.
The concentrations of LTB4 in the supernatant fluids in
response to ACh and SP at the concentrations of 100 µM for 24-h
incubation were significantly increased compared with control (Fig.
7, P < 0.01). In contrast, PAF
in BBEC supernatant fluids in control and in response to ACh and SP was
not detected.
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Fig. 7.
Release of LTB4 from bovine bronchial epithelial cells in
response to ACh and SP (n = 6 experiments). Values are means ± SE. * P < 0.01 compared with control (Cont).
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| |
DISCUSSION |
Many eosinophil chemotactic factors are released from a variety of
cells. Recently, cytokines, including granulocyte-macrophage colony-stimulating factor; Regulated on Activation Normal T cells, Expressed and Secreted; and interleukin (IL)-3, IL-4, IL-5, and IL-8,
have been reported to play roles in eosinophilopoesis and differentiation (11, 32). LTB4, a potent inflammatory cell chemotactic factor, can be released from a variety of cells, including neutrophils, eosinophils, alveolar macrophages (34), and mononuclear cells, and is present in the BALF recovered from asthmatic patients (1). PAF, platelet factor 4, activated complement, and
fibrin-degradation product have also been suggested to play a role in
eosinophil recruitment observed in asthma (9, 12, 23). Two
tetrapeptides and histamine have also been suggested as eosinophil
chemotactic factors (14). However, the participation of the airway cell in the recruitment of eosinophils and the role of neuroregulation for
the ECA release are unknown. In the present study, BBEC released ECA in
response to ACh and SP in a dose- and time-dependent fashion. The
released activity was chemotactic, as certified by checkerboard analysis. Partial characterization revealed that the activity was
predominantly lipid extractable. The column chromatography showed that
the lowest molecular mass chemotactic peak was that of predominant
activity. Both the 5-lipoxygenase inhibitor and LTB4-receptor antagonist blocked ECA. Finally, the release
of LTB4 was significantly increased in response to ACh and
SP, reaching a level that induced eosinophil chemotaxis. These data
suggest that neuroregulation of ECA release from the airway cells may play a role in eosinophil recruitment into the airway.
SP and ACh are thought to be a potential neuroregulator in bronchial
asthma (3). These substances have direct effects on airway functions,
including the increases in airway hyperreactivity, airway smooth muscle
cell contraction, and airway secretion (3, 4). On the other hand,
several lines of evidence support the concept that eosinophil
infiltration may lead to an increase in airway hyperreactivity. In
patients with asthma, the disease severity correlates with the degree
of bronchial wall infiltration of eosinophils (6). The study of BALF
demonstrates a correlation between the presence and activity of
eosinophils and the severity of the asthmatic disease (6). Patients who
developed a late asthmatic reaction have a raised number of blood
eosinophils and a raised eosinophil number in BALF (8). Thus the
release of ECA from BBEC in response to ACh and SP suggests a role for
the airway cells in the increase of eosinophil
infiltration in the bronchial wall and lumen in patients with asthma
and may suggest the possibility of neuroregulation of the eosinophil
recruitment, in addition to the known neural regulation of the airway
functions.
The released chemotactic activity from BBEC in the present study is not
yet completely characterized. However, the predominant ECA was lipid
extractable. The nonspecific and 5-specific lipoxygenase inhibitors
blocked the release of chemotactic activity, suggesting that the
activity may be derived from the 5-lipoxygenase pathway. The lowest
molecular mass peak ECA was blocked by LTB4-receptor antagonist. Finally, immunoreactive LTB4 in the supernatant
fluids increased to a level that was chemotactic for eosinophils. On the basis of these lines of evidence, LTB4 is the
predominant ECA. In support of this concept is the fact that
LTB4 and 12-lipoxygenase-pathway products can be released
from bovine airway cells (17, 26, 27), and LTB4 and
15-dihydroxyeicosatetraenoic acid are released from dog (24) and human
tracheal epithelial cells (22), respectively. These lipoxygenase
metabolites are chemotactic for eosinophils (1, 15, 24, 33). The
release of lipoxygenase metabolites is species specific and differs in
response to a variety of stimuli, even in the same species. However, it
has many common biological characteristics. Thus neuroregulation of the
airway cells may play a role in the release of lipoxygenase-derived
ECA.
The ethyl acetate extraction and trypsin digestion of the supernatant
fluids resulted in the increase in chemotactic activity, compared with
the crude sample. Although the mechanisms underlying this potentiation
of ECA are unknown, the presence of eosinophil-migration inhibitor in
the supernatant fluids may be one of the possible explanations. The
removal by ethyl acetate extraction or destruction by trypsin of
eosinophil-migration inhibitor resulted in the potentiation of ECA. If
this is the case, this inhibitory factor(s) may prolong eosinophil
retention in the bronchial lumens.
In conclusion, BBEC released ECA in response to ACh and SP in a dose-
and time-dependent fashion. The released activity was chemotactic, as
shown by checkerboard analysis and low-molecular-mass lipoxygenase-derived activity, i.e., LTB4. The results may
suggest the possibility of a role for neuroregulation of the eosinophil recruitment in the bronchial wall and lumen, in addition to the known
neural regulation of the airway functions.
| |
FOOTNOTES |
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 15 January 1997; accepted in final form 30 December 1997.
| |
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