Effects of eosinophil granule major basic protein on
phosphatidylcholine secretion in rat type II pneumocytes
Manabu
Okumura1,2,
Hirofumi
Kai2,
Shinya
Shinozawa1,
Yoichiro
Isohama2, and
Takeshi
Miyata2
1 Department of Pharmacy,
Miyazaki Medical College, Miyazaki 889-1692; and
2 Department of Pharmacological
Sciences, Faculty of Pharmaceutical Sciences, Kumamoto University,
Kumamoto 862, Japan
 |
ABSTRACT |
Eosinophils are involved in inflammatory
diseases such as asthma. We previously reported that activated
eosinophils increased the phosphatidylcholine (PC) secretion in primary
cultures of rat type II pneumocytes. Increased PC secretion was
confirmed to be partly mediated by superoxide anions released from
activated eosinophils. However, the influence of eosinophil granule
proteins on PC secretion is unknown at present. In this study, we
determined whether eosinophil major basic protein (MBP) influences PC
secretion. MBP dose dependently increased the PC secretion in rat type
II pneumocytes without producing any cell damage. The MBP-induced increase in PC secretion was significantly reduced by preadministration of either H-7, a protein kinase inhibitor, or
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM, a chelator of intracellular
Ca2+, but not by H-89, a protein
kinase inhibitor. Our results suggest that the MBP-induced increase in
PC secretion may provide mechanical stability and protect against lung atelectasis.
asthma; surfactant
 |
INTRODUCTION |
EOSINOPHILS PLAY an important pathological role in
allergic diseases such as asthma, and examination of bronchoalveolar
lavage fluid of asthmatic patients usually demonstrates a significant increase in eosinophil count (19, 29). Eosinophils release several
particles including leukotrienes; platelet-activating factor; various
oxygen-derived toxic metabolites such as superoxide anions,
H2O2,
and hydroxyl radicals (28); and toxic cationic proteins such as major
basic protein (MBP), eosinophil cationic protein (ECP), eosinophil
peroxidase (EPO) and eosinophil-derived neurotoxin (EDN) (9, 11). The
cytotoxic proteins and other mediators cause hyperreactivity of
respiratory smooth muscles (7) as well as desquamation of and damage to
respiratory epithelial cells including type II pneumocytes.
Type II pneumocytes produce lung surfactant to reduce the surface
tension of the alveolar air-liquid interface, thereby providing mechanical stability and preventing alveolar atelectasis (2). Hence
examination of the influence of cytotoxic proteins and other mediators
released from eosinophils on the secretion of lung surfactant is
important for our understanding of normal lung physiology as well as of
certain pathological pulmonary conditions. Previous studies (2, 15, 21)
have shown an increased secretion of phosphatidylcholine (PC), the
predominant component of pulmonary surfactant, by a variety of
physiological and pharmacological agents. Furthermore, a recent study
by Okumura et al. (22) showed that activated eosinophils
increase PC secretion in primary cultures of rat type II pneumocytes.
Such an increase was not suppressed by ONO-1078, a selective antagonist
of peptide leukotrienes, or TCV-309, an antagonist of
platelet-activating factor, but was suppressed by a combination of
superoxide dismutase and catalase. However, our results also showed
that the combined use of both enzymes did not completely inhibit the
secretion of PC. These results suggested that increased PC secretion
was partly mediated by superoxide anions released from activated
eosinophils and might represent one facet of the defense mechanisms
aimed at attenuating cellular damage induced by superoxide anions.
Superoxide anions released from eosinophils participate in the early
asthmatic reaction, whereas the eosinophil granule proteins participate
in the late asthmatic reactions (LAR). Eosinophil granule proteins
increase the secretion of histamine by basophils and mast cells (30)
and the generation of superoxide anion by lung macrophages (14).
However, to our knowledge, the effect of eosinophil granule proteins on
the secretion of lung surfactant has not been previously reported.
Based on the above findings and the results of the previous study by
Okumura et al. (22) demonstrating failure of the combined use of superoxide dismutase and catalase to completely inhibit the
activated eosinophil-induced increase in PC secretion, we hypothesized
in the present study that eosinophil granule proteins might increase
the secretion of lung surfactant in patients with asthma during LAR. To
test this hypothesis, we examined the effects of MBP, a primary
constituent of eosinophil granule proteins, on PC secretion in primary
cultures of rat type II pneumocytes.
 |
MATERIALS AND METHODS |
Animals and chemicals. Rats and guinea
pigs were purchased from Kyudo Farm (Fukuoka, Japan), tissue culture
medium was from Nissui Pharmaceutical (Tokyo, Japan), and fetal bovine
serum was from JRH Bioscience (Lenexa, KS).
[Methyl-3H]choline
and Aquasol II were obtained from NEN Research Products (Boston, MA).
Sephadex G-50 was obtained from Pharmacia Fine Chemicals (Uppsala,
Sweden). H-7, H-89, and HA-1004 were purchased from Seikagaku (Tokyo,
Japan).
1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM was from Wako Pure Chemical Industries (Osaka, Japan).
Other reagents and biochemicals were purchased from Sigma (St. Louis,
MO). The experimental protocol was approved by the Ethics Review
Committees for Animal Research of Miyazaki Medical College (Miyazaki,
Japan) and the Faculty of Pharmaceutical Sciences, Kumamoto University
(Kumamoto, Japan).
Primary cultures of rat type II
pneumocytes. Type II pneumocytes were isolated from the
lungs of adult specific pathogen-free male Wistar rats (body weight
180-200 g) according to the method of Dobbs et al. (4). This
method yields ~107 cells/rat.
Cells were suspended at 106
cells/ml in Dulbecco's modified Eagle's medium supplemented with 10%
fetal bovine serum, 74 kBq/ml of
[methyl-3H]choline
(specific activity 3.0 TBq/mmol), 100 U/ml of penicillin, and 100 µg/ml of streptomycin; plated on 24-well tissue culture plates (Falcon); and then cultured at 37°C in 5%
CO2-air for 18 h. Nonadherent
cells were removed from the wells by washing before the assay. For
cellular identification, the sample was stained with tannic
acid-polychrome stain (17) and alkaline phosphatase stain (6). The
purity of the type II pneumocytes was 95 ± 7% (SE;
n = 8 cultures), and their viability
was 97 ± 8% (n = 8 cultures) as
confirmed by the trypan blue exclusion test.
Preparation of guinea pig MBP.
Eosinophils were isolated from peritoneal exudates of Hartley guinea
pigs by a modification of the method originally described by Pincus
(24). Briefly, peritoneal eosinophil-rich exudates were produced by
weekly intraperitoneal injections of 7,500 U/ml of polymyxin B sulfate
for at least 8 wk. Furthermore, 50 ml of Hanks' balanced salt solution
containing 20 U/ml of sodium heparin were injected intraperitoneally
the day after the last injection of polymyxin B sulfate. The abdomen was massaged gently, and the peritoneal fluid was collected. The fluid
was fractionated by centrifugation through solutions of Nycodenz. The
purified fraction of eosinophils was collected from the interface
between 1.088 and 1.098 g/ml in the gradient. Eosinophils were 96%
pure as determined with Litt's stained smears. MBP was purified from
isolated guinea pig eosinophils according to the procedure described by
Gleich et al. (11). Briefly, suspensions of eosinophils in 0.34 M
sucrose were repeatedly pipetted, then centrifuged for 10 min at 400 g at 4°C to remove unbroken cells. The opalescent supernatants were transferred to another tube and centrifuged at 25,000 g for 20 min at
4°C. The pellet was solubilized in 0.02 M acetate buffer, pH 4.3, in 0.01 M HCl and analyzed by gel filtration on 1.2 × 45-cm
columns of Sephadex G-50. The purified MBP was homogeneous as confirmed
by the presence of a single electrophoretic protein band on SDS-PAGE
gels as described previously (10; data not shown). The concentration of
MBP was determined as described previously (10, 12) and was adjusted to
100-fold of the indicated final concentration with sodium acetate
buffer. Sodium acetate buffer did not alter spontaneous PC secretion.
Metabolic labeling of PC and treatment of
cultures. Secretion of PC by cultured type II
pneumocytes was determined as follows. The cells were rinsed with fresh
serum- and antibiotic-free medium to remove
[methyl-3H]choline
and unattached cells. MBP was added 30 min after the rinse, followed by
incubation for 90 min. At the end of the incubation period, the medium
was aspirated, the cells were lysed with 2 ml of ice-cold 0.05% Triton
X-100 solution, and the lipids were extracted from both the cells and
medium with chloroform-methanol (2:1 vol/vol). PC was separated from
other phospholipids by thin-layer chromatography (18), and its
radioactivity was measured with a liquid scintillation counter after
the addition of 5 ml of Aquasol II to each sample. Secretion is
expressed as the amount of
[3H]PC in the medium
after a 90-min incubation as a percentage of that in the cells plus medium.
Detection of cell membrane damage. The
presence or absence of cytoplasmic leakage due to cell membrane damage
after MBP treatment was determined by measuring lactate dehydrogenase
(LDH) activity in the culture medium with a commercial LDH assay kit
(Nippon Shoji). LDH activity released into the medium did not exceed
1% of the total cell content in all experiments (data not shown).
Statistical analysis. The
concentration of PC is expressed as mean ± SE. Differences among
groups were assessed by Duncan's multiple range test (a nonparametric
test). P < 0.05 denoted the presence
of a significant difference.
 |
RESULTS |
Isolation of eosinophil granule proteins and their
effect on PC secretion. Similar to a previous study on
eosinophil granule proteins of guinea pigs (30), suspensions containing
eosinophil granule proteins (EPO, EDN, ECP, and MBP) showed three
separate peak fractions by gel filtration on columns of Sephadex G-50
when each fraction was monitored for changes in absorbance at 277 nm (Fig. 1,
top). The first peak
(G2) corresponded to the void volume of the column and contained a variety of bands as observed on SDS-PAGE
gels. The second peak (G3) contained
two slightly separated bands, whereas the third peak
(G4) contained one major band with a
molecular weight < 14,000. These results indicated that the third
peak contained only MBP.

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Fig. 1.
Top: fraction profile of eosinophil
granule protein by gel filtration on columns of Sephadex G-50. Each
fraction was monitored for changes in absorbance at 277 nm.
G1, fraction preceding void volume;
G2, eosinophil peroxidase (EPO)
fraction; G3, eosinophil cationic
protein (ECP) and eosinophil-derived neurotoxin (EDN) fraction;
G4, major basic protein (MBP).
Bottom: effects of each fraction on PC
secretion in primary cultures of rat type II pneumocytes. Each fraction
was ultrafiltered, concentrated, and resuspended at a granule
concentration corresponding to 1 × 106 eosinophils/ml culture medium.
Values are means ± SE expressed as amount of
[3H]phosphatidylcholine
(PC) in medium after 90-min incubation as percentage of that in cells
plus medium (% of control) from 6 experiments.
[3H]PC secretion after
90 min was 0.75 ± 0.07% in control cultures
(n = 5). Significant difference from
control value: * P < 0.05;
** P < 0.01.
|
|
Next, we examined the effects of various peak fractions, including the
fraction preceding the void volume
(G1), on PC secretion in primary
cultures of rat type II pneumocytes. Each fraction was ultrafiltered,
concentrated, and resuspended at a granule concentration corresponding
to 1 × 106 eosinophils/ml
culture medium. G1 and
G3 fractions did not increase PC
secretion; in contrast, G2 and
G4 fractions significantly increased PC secretion by 21 and 54%, respectively (Fig. 1,
bottom).
Effects of MBP on PC secretion. MBP
caused an ~1.5-fold increase in PC secretion in primary cultures of
rat type II pneumocytes without increasing LDH activity in the culture
medium. The MBP-induced increase of PC secretion was concentration
dependent, and the peak secretion was observed at an MBP concentration
of 8 × 10
9 M (Fig.
2). Furthermore, examination of the
kinetics of PC secretion showed that it commenced within 5 min of the
addition of MBP (8 × 10
9 M) and then increased
progressively throughout the observation period (90 min; Fig.
3). The profile was not
different from that of the control culture. In contrast, the addition
of terbutaline (1 × 10
6 M), which was used as
protein kinase A-related agent, resulted in a steep increase in the
first 30 min, but this was followed by a small increase throughout the
remaining 60 min (Fig. 3).

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Fig. 2.
MBP concentration-dependent secretion of PC from type II pneumocytes.
Isolated pneumocytes were incubated with indicated concentrations of
MBP for 90 min. , Value for terbutaline (1 × 10 6 M; positive control).
Values are means ± SE from 6 experiments.
[3H]PC secretion after
90 min was 0.51 ± 0.04% in control cultures (not incubated with
MBP; n = 5).
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Fig. 3.
Kinetics of MBP-stimulated PC secretion in type II pneumocytes. ,
Control culture; , MBP (8 × 10 9 M); , terbutaline (1 × 10 6 M). Values are
means ± SE from 6 experiments.
[3H]PC secretion after
90 min was 0.89 ± 0.03% in control cultures
(n = 5).
|
|
Effects of several inhibitors on MBP-induced increase
in PC secretion. To determine the mechanism involved in
MBP-induced increase in PC secretion, we examined the effects of
several inhibitors of intracellular pathways. In these experiments, the
inhibitor was added 10 min before the application of 8 × 10
9 M MBP. H-7 (1 × 10
5 M), a protein kinase C
inhibitor, and BAPTA-AM (5 × 10
6 M), a chelator of
intracellular Ca2+, significantly
suppressed the MBP-induced increase in PC secretion. However, no
synergistic effect was noted when the two inhibitors were added
simultaneously. H-89 (6 × 10
6 M), a protein kinase A
inhibitor, and HA-1004 (1 × 10
5 M), a control to H-7,
did not influence the MBP-induced increase in PC secretion.
 |
DISCUSSION |
The major finding of the present study was that MBP purified from
guinea pig eosinophil granules increased PC secretion in primary
cultures of rat type II pneumocytes and that such increases were MBP
concentration dependent (Fig. 2). Furthermore, we also demonstrated
that MBP-stimulated PC secretion was significantly inhibited by H-7, a
protein kinase inhibitor, and BAPTA-AM, an intracellular
Ca2+ chelator (Fig.
4).

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Fig. 4.
Effects of several inhibitors on MBP-induced PC secretion in type II
pneumocytes. Each inhibitor was added 10 min before addition of MBP (8 × 10 9 M), followed by
further incubation for 90 min. H-89 (6 × 10 6 M), HA-1004 (1 × 10 5 M), H-7 (1 × 10 5 M), and
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid (BAPTA)-AM (5 × 10 6 M) were used as
inhibitors of PC secretion pathways. -, In presence of MBP alone.
Values are means ± SE from 6 experiments.
[3H]PC secretion after
90 min was 0.88 ± 0.06% in control cultures
(n = 5). * Significant
difference from MBP alone, P < 0.05.
|
|
Eosinophils infiltrate the airways and lungs of asthmatic patients and
release many granule proteins, leukotrienes, platelet-activating factor, and various oxygen-derived toxic metabolites. The direct involvement of these substances on lung surfactant has been reported (8, 25). However, the effect of the eosinophil itself on the secretion
of surfactant is poorly understood. In a previous study by Okumura et
al. (22), activated eosinophils were found to increase PC secretion in
primary cultures of rat type II pneumocytes, which was partly mediated
by superoxide anion released from these cells. However, we speculated
that the granule proteins, in addition to superoxide anions, were
probably involved in the increased PC secretion because such secretion
was not completely inhibited by pretreatment with superoxide dismutase
combined with catalase. To our knowledge, the possible involvement of
eosinophil granule proteins on PC secretion has not been previously investigated.
Previous studies (13, 20) have shown that MBP causes desquamation of
and damage to respiratory epithelial cells. Furthermore, MBP has been
reported to stimulate histamine release from human basophils and rat
mast cells (30). Histamine increases the surfactant secretion by a
receptor-mediated process (3). Histamine release is an important
pathophysiological reaction in asthma, and the increased surfactant
secretion induced by histamine may be one of the protective reactions
during the early asthmatic reaction. However, our results showed that
the concentration of MBP necessary to increase PC secretion was far
less than that of MBP to stimulate histamine release. Our results
suggest that secretion of lung surfactant may be increased in asthmatic
patients by granule proteins released from eosinophils during the early
stages of the LAR. Such an increase in pulmonary surfactant secretion
may serve as a protective mechanism against cellular damage caused by
cytotoxic granule proteins.
MBP is the primary constituent of eosinophil granules (10, 12) and
accounts for >50% of the granule proteins in the guinea pig (11,
16). Furthermore, previous studies have shown high serum concentrations
of MBP in patients with eosinophilia (1) and in the sputum and
bronchoalveolar lavage fluid of asthmatic patients (5, 29). Based on
these early findings, we examined, in the present study, the specific
effects of MBP. In addition to MBP, eosinophil granules contain three
other major cationic proteins, EPO, EDN, and ECP, that have been
purified from peritoneal exudates in the guinea pig. In this study,
these granule proteins were separated from each other by Sephadex G-50
as described in a previous study (30). The first protein peak contained
EPO, and the second peak contained EDN and ECP. The pooled fraction containing EPO increased PC secretion significantly, but the
concentration of EPO was lower than that of MBP. In contrast, the
pooled fraction containing EDN and ECP did not increase PC secretion.
These results suggested that in addition to MBP, EPO may also
participate in increasing PC secretion induced by eosinophils in
primary cultures of rat type II pneumocytes. However, taking into
consideration the relatively low amount of EPO, our results show that
the majority of secreted PC was mediated to a large extent by MBP.
PC secretion from type II pneumocytes is regulated via various
intracellular pathways (2, 15, 21, 23). The principal secretion pathway
is the activation of a cAMP-dependent protein kinase, protein kinase C,
and high concentrations of intracellular Ca2+. In basophils, MBP stimulates
histamine release, which is inhibited by pertussis toxin (27). Also, in
mast cells, histamine release is induced by protein kinase C activation
by diacylglycerol through a pertussis toxin-sensitive G protein. In
type II pneumocytes, H-7, but not HA-1004, significantly but partly
inhibited the MBP-increased PC secretion. H-7 is a relatively selective
protein kinase C inhibitor, but it also exerts a slight inhibitory
effect on protein kinase A and protein kinase G as well as on
Ca2+/calmodulin-dependent kinase.
In contrast, the HA-1004 dose not have any inhibitory effect on protein
kinase C, although it is inhibitory of other kinases, with a potency
equivalent to that of H-7. Therefore, MBP may stimulate PC secretion as
well as histamine release in basophils and mast cells through protein
kinase C activation. In support of this, the kinetics of MBP-stimulated
PC secretion (Fig. 3) is similar to that of
1-oleoyl-2-acetyl-sn-glycerol-stimulated PC secretion previously reported (26). BAPTA-AM also significantly inhibited PC secretion increased by MBP, suggesting that intracellular Ca2+ plays an important role in
MBP stimulation. The finding that BAPTA-AM in combination with H-7 did
not synergistically or additively inhibit MBP-induced PC secretion
(Fig. 4) suggests that intracellular Ca2+ and protein kinase C may act
on the same signaling pathway of MBP stimulation of PC secretion.
Furthermore, incomplete inhibition of PC secretion by BAPTA-AM and H-7
suggests that other pathways may be involved in MBP-induced PC
secretion, although further studies with higher concentrations of H-7
and other protein kinase C inhibitors are needed.
In conclusion, our results showed that MBP, the primary constituent of
eosinophil granule proteins, increased PC secretion in primary cultures
of rat type II pneumocytes. Such an effect of eosinophils on PC, the
predominant component of pulmonary surfactant, may provide mechanical
stability and prevent lung atelectasis in asthmatic patients during
early LAR. Our results also showed that the MBP-induced increase in PC
secretion was mediated, at least in part, by protein kinase C and
intracellular Ca2+. Further
studies are necessary to identify other pathways that regulate PC secretion.
 |
ACKNOWLEDGEMENTS |
This work was supported by Grants-in-Aid for Scientific Research
from the Ministry of Education, Science and Culture, Japan.
 |
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: T. Miyata, Dept.
of Pharmacological Sciences, Faculty of Pharmaceutical Sciences,
Kumamoto Univ., 5-1 Oe-Honmachi, Kumamoto 862, Japan. (E-mail:
tmiyata{at}gpo.kumamoto-u.ac.jp).
Received 25 August 1998; accepted in final form 19 January 1999.
 |
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