Vol. 284, Issue 5, L720-L726, May 2003
A potent inhibitor of cytosolic phospholipase A2,
arachidonyl trifluoromethyl ketone, attenuates LPS-induced lung
injury in mice
Takahide
Nagase1,
Naonori
Uozumi2,
Tomoko
Aoki-Nagase1,
Kan
Terawaki2,3,
Satoshi
Ishii2,
Tetsuji
Tomita1,
Hiroshi
Yamamoto1,
Kohei
Hashizume3,
Yasuyoshi
Ouchi1, and
Takao
Shimizu2,4
Departments of 1 Geriatric Medicine,
2 Biochemistry and Molecular Biology, and
3 Pediatric Surgery, Graduate School of Medicine,
University of Tokyo, and 4 Core Research for
Evolutional Science and Technology of Japan Science and Technology
Corporation, Tokyo 113, Japan
 |
ABSTRACT |
Acute respiratory distress syndrome
(ARDS) is an acute lung injury of high mortality rate, and sepsis
syndrome is one of the most frequent causes of ARDS. Metabolites of
arachidonic acid, including thromboxanes and leukotrienes, are
proinflammatory mediators and potentially involved in the development
of ARDS. A key enzyme for the production of these inflammatory
mediators is cytosolic phospholipase A2
(cPLA2). Recently, it has been reported that arachidonyl
trifluoromethyl ketone (ATK) is a potent inhibitor of
cPLA2. In the present study, we hypothesized that
pharmacological intervention of cPLA2 could affect acute
lung injury. To test this hypothesis, we examined the effects of ATK in
a murine model of acute lung injury induced by septic syndrome. The
treatment with ATK significantly attenuated lung injury,
polymorphonuclear neutrophil sequestration, and deterioration of
gas exchange caused by lipopolysaccharide and zymosan administration.
The current observations suggest that pharmacological intervention of
cPLA2 could be a novel therapeutic approach to acute lung
injury caused by sepsis syndrome.
acute respiratory distress syndrome; lipopolysaccharide; sepsis; eicosanoid; leukotriene
| |
INTRODUCTION |
ACUTE RESPIRATORY
DISTRESS SYNDROME (ARDS) is characterized by acute lung injury,
and severe sepsis is one of the most important causes of ARDS (9,
10, 31). Although patients with ARDS are intensively treated
with currently available drugs, the mortality rate for ARDS remains
high, and it ranges from 40 to 70%. Potential mechanisms that
cause ARDS include damage to the alveolar-capillary membrane and
polymorphonuclear neutrophil (PMN) adhesion, activation, and
sequestration, leading to respiratory failure (9, 10, 31).
Platelet-activating factor (PAF) and metabolites of arachidonic acid
are potentially involved in the development of ARDS (23, 30,
32). PAF is a proinflammatory mediator produced from
phospholipids (12-14). Thromboxanes (TXs) and
leukotrienes (LTs) are potent mediators generated from arachidonic acid
by cyclooxygenase and 5-lipoxygenase (7), respectively.
TXA2 may increase lung permeability, whereas LTB4 is a potent neutrophil chemoattractant. Phospholipase
A2 (PLA2) is a key enzyme for the production of
proinflammatory mediators, including eicosanoids and PAF. Although a
number of distinct types of PLA2 have been reported to be
characterized, cytosolic PLA2 (cPLA2) is
thought to be particularly important (29, 30, 34, 36, 37).
The cPLA2 preferentially hydrolyzes phospholipids containing arachidonic acid and is activated by submicromolar concentration of Ca2+ and by phosphorylation of a serine
residue (5, 16, 18, 33). Recently, it has been reported
that an analog of arachidonic acid in which the 
COOH functionality is
replaced by COCF3, named arachidonyl trifluoromethyl
ketone (ATK), is a potent and selective slow-binding inhibitor of
cPLA2 (33, 35).
In the present study, we hypothesized that pharmacological intervention
of cPLA2 could affect acute lung injury. To test this hypothesis, we chose to use ATK as an inhibitor of cPLA2
and examined the effects of ATK in a murine model of acute lung injury
induced by lipopolysaccharide (LPS) and zymosan administration.
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METHODS |
Animal preparation.
We used male C57BL/6 mice (7-8 wk old). Animals were anesthetized
with pentobarbital sodium (25 mg/kg ip) and ketamine hydrochloride (25 mg/kg ip) in combination and then paralyzed with pancuronium bromide
(0.3 mg/kg ip). Anesthesia and paralysis were maintained by
supplemental administration of 10% of the initial dose every hour.
After tracheostomy, a metal endotracheal tube (inside diameter 1 mm,
length 8 mm) was inserted in the trachea. Animals were mechanically ventilated (model 683; Harvard Apparatus, South Natick, MA) with tidal
volumes of 10 ml/kg and frequencies of 2.5 Hz. We opened the thorax
widely by means of midline sternotomy and applied a positive end
expiratory pressure of 2 cmH2O by placing the expired line
underwater. During the experiments, oxygen gas was continuously supplied to the ventilatory system
(FIO2 = 1.0). A heating pad was used
to maintain the body temperature of animals. To assess the development
of lung injury physiologically, we measured lung elastance
(EL) and resistance (RL) as
previously described (1, 21-27). Briefly, we measured
the tracheal pressure (Ptr), flow, and volume (V). We calculated
EL and RL by adjusting the equation of motion: Ptr = EL · V + RL(dV/dt) + K, where
K is a constant. Changes in EL and
RL reflect lung parenchymal alterations and stiffening of the lungs.
Experimental acute lung injury induced by LPS/zymosan
administration.
One minute before intravenous administration, two deep inhalations
(three times tidal volume) were delivered to standardize volume history
and measurements were made as baseline. In the physiological study,
mice were divided into four experimental groups, i.e., saline-treated
(n = 6), ATK/saline-treated (n = 4),
LPS/zymosan-treated (n = 7), and
ATK/LPS/zymosan-treated groups (n = 5). In the
LPS/zymosan-treated group, mice received 3 mg/kg LPS from
Escherichia coli O111:B4 (Sigma Chemical, St. Louis, MO)
intravenously. Two hours later, 10 mg/kg of zymosan A from Saccharomyces cerevisiae (Sigma) were intravenously
administered (19, 30). In the saline-treated group,
animals received saline instead of LPS and zymosan in the same manner
and served as controls. In the ATK-treated group, 20 mg/kg ATK (Cayman
Chemical, Ann Arbor, MI) were administered intraperitoneally 30 min
before saline or LPS administration. The current dose of ATK and timing
of ATK administration were applied on the basis of previous reports
(11, 20) and our preliminary experiments. In all groups,
measurements were made at 30-min intervals for 4 h.
Assessment of respiratory failure.
At the end of experiment, blood samples for gas analysis were obtained
from the left ventricle. We then measured PaO2,
PaCO2, and pH to assess the extent of respiratory
failure (blood gas analyzer; AVL Medical Systems, Schaffhausen, Switzerland).
Bronchoalveolar lavage fluid.
At the end of the experiment, bronchoalveolar lavage (BAL) was
performed (1 ml of phosphate-buffered saline five times) in saline-treated (n = 6), LPS/zymosan-treated
(n = 7), and ATK/LPS/zymosan-treated groups
(n = 5). In each animal, 90% (4.5 ml) of the total
injected volume was consistently recovered. After BAL fluid (BALF) was centrifuged at 450 g for 10 min, total and differential cell
counts of the BALF were determined from the cell fraction (29,
30). The supernatant was stored at 70°C until measurement of
protein was performed. The concentration of protein was measured by
Lowry's method with bovine serum albumin as a standard.
TX and LT assay.
TXA2 (measured as TXB2), LTB4, and
LTC4/D4/E4 in the BALF were
determined by enzyme immunoassay (EIA) kits (Amersham Pharmacia Biotech, Piscataway, NJ). The detection limits of the EIA assays for
TXB2, LTB4, and
LTC4/D4/E4 were 3.6, 6, and 10 pg/ml, respectively.
Myeloperoxidase activity assay.
At the end of experiments, the left lungs were removed from
mice of each group (n = 4, respectively).
Myeloperoxidase (MPO) activity was measured as previously reported
(3, 15). Briefly, the frozen lungs were weighed and
homogenized in hexadecyltrimethylammonium bromide (HTAB) buffer (0.5%
HTAB in 50 mM phosphate buffer, pH 6.0). The homogenates were sonicated
and centrifuged at 40,000 g for 15 min. The supernatant was
mixed with assay buffer containing potassium phosphate buffer,
H2O2, and o-dianisidine
hydrochloride. Then, the supernatant was placed in a spectrophotometer
for reading at 460 nm as previously described (3, 15).
Histological study.
At the end of experiments, the right lungs of the mice were removed and
fixed with 10% formalin. After fixation, the tissue blocks obtained
from midsagittal slices of the lungs were embedded in paraffin. Blocks
were cut 4 µm thick with a microtome, and then hematoxylin-eosin
staining was performed.
Data analysis.
Comparisons of data among each experimental group were carried out with
analysis of variance. If statistical significances were detected, a
Scheffé test was then applied as a post hoc test. Data are
expressed as means ± SE. P values <0.05 were taken as significant.
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RESULTS |
Physiological data following LPS/zymosan or saline administration.
There were no significant differences in baseline EL and
RL among each group. Fig.
1 and Table
1 demonstrate the physiological data
after LPS/zymosan or saline administration. As shown, EL and RL in LPS/zymosan-treated group were
significantly increased compared with saline-treated group, which
reflects physiological alterations in lung parenchyma. The
administration of ATK significantly reduced LPS/zymosan-induced
responses in EL and RL, whereas
there were significant differences between saline-treated and
ATK/LPS/zymosan-treated groups.
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Fig. 1.
The time course of response in lung elastance in
saline-treated (SAL, n = 6), arachidonyl
trifluoromethyl ketone (ATK)/saline-treated (SAL+ATK,
n = 4), LPS/zymosan-treated (LPS/Z, n = 7), and ATK/LPS/zymosan-treated groups (LPS/Z+ATK, n = 5). In LPS/zymosan-treated groups, zymosan was administered 2 h
after LPS treatment, whereas saline was treated in the same fashion in
the saline-treated groups. *P < 0.001 vs.
saline-treated group; #P < 0.001 vs.
LPS/zymosan-treated group.
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Administration of LPS/zymosan elicited respiratory failure, which was
not observed in saline-treated groups. Hypoxemia was prominent in
LPS/zymosan-treated mice, whereas ATK administration reduced
LPS/zymosan-induced hypoxemia (Fig. 2).
After LPS/zymosan treatment, increases in PaCO2 and
decreases in pH were observed, although there were no differences in
PaCO2 or pH levels between saline-treated and
ATK/LPS/zymosan-treated groups. As shown, ATK had little effect on
physiological data in saline-treated groups.
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Fig. 2.
Effects of cytosolic phospholipase A2
(cPLA2) inhibitor ATK in hypoxemia induced by LPS/zymosan
treatment. *P < 0.001 vs. saline-treated group;
#P < 0.001 vs. LPS/zymosan-treated group.
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Analyses of BALF.
Table 2 and Figs. 3 and 4 summarize the
analyzed data of BALF. As shown,
LPS/zymosan administration increased
protein amount and number of PMN in BALF, indicating LPS/zymosan
induced protein leakage and PMN infiltration. The protein leakage and
PMN sequestration were significantly attenuated by the treatment of
ATK. Meanwhile, there were significant differences in BALF protein
amount and number of PMN between saline-treated and
ATK/LPS/zymosan-treated groups.
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Fig. 3.
Effects of cPLA2 inhibitor ATK in protein
leakage induced by LPS/zymosan treatment. BALF, bronchoalveolar lavage
fluid. *P < 0.01 vs. saline-treated group;
#P < 0.01 vs. LPS/zymosan-treated group.
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Fig. 4.
Effects of cPLA2 inhibitor ATK in neutrophil
infiltration induced by LPS/zymosan treatment. PMN, polymorphonuclear
neutrophil. *P < 0.001 vs. saline-treated group;
#P < 0.001 vs. LPS/zymosan-treated group.
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TX and LT assay.
To assess the biosynthesis of cPLA2 products, we performed
TXA2 (measured as TXB2), LTB4, and
LTC4/D4/E4 assay of the BALF. Figures 5-7 summarize the results of
BALF TXB2,
LTB4, and LTC4/D4/E4 assay in each experimental group.
LPS/zymosan administration markedly increased TXB2,
LTB4, and LTC4/D4/E4
levels in BALF compared with the saline-treated group, whereas the
levels of these eicosanoids were significantly reduced in the
ATK/LPS/zymosan-treated group. However, there were significant
differences in BALF TXB2, LTB4, and
LTC4/D4/E4 levels between
saline-treated and ATK/LPS/zymosan-treated groups.
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Fig. 5.
Effects of cPLA2 inhibitor ATK in thromboxane
(TX) B2 production induced by LPS/zymosan treatment.
*P < 0.05 vs. saline-treated group; #P < 0.01 vs. LPS/zymosan-treated group.
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Fig. 6.
Effects of cPLA2 inhibitor ATK in
LTB4 production induced by LPS/zymosan treatment.
*P < 0.001 vs. saline-treated group;
#P < 0.05 vs. LPS/zymosan-treated group.
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Fig. 7.
Effects of cPLA2 inhibitor ATK in leukotriene
(LT) C4/D4/E4 production induced by
LPS/zymosan treatment. *P < 0.001 vs. saline-treated
group; #P < 0.05 vs. LPS/zymosan-treated group.
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MPO activity assay.
To assess the PMN infiltration in the lung, we performed MPO activity
assay. Figure 8 shows the results of MPO
activity in lung tissue. LPS/zymosan administration markedly increased
MPO activity in lungs compared with the saline-treated group, whereas the MPO activity was significantly attenuated in the
ATK/LPS/zymosan-treated group. However, no significant difference in
lung MPO activity was observed between saline-treated and
ATK/LPS/zymosan-treated groups.
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Fig. 8.
Effects of cPLA2 inhibitor ATK in lung
myeloperoxidase (MPO) activity induced by LPS/zymosan treatment
(n = 4 for each group). *P < 0.05 vs.
saline-treated group; #P < 0.05 vs.
LPS/zymosan-treated group.
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Histological study.
Figure 9 represents lung histology
following LPS/zymosan administration. As shown, LPS/zymosan
administration induced prominent lesions, as well as alveolar
thickening, distortion, and cellular infiltration. In contrast,
the alveolar architecture is well preserved and histological changes
are minimal in ATK-treated animals.
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Fig. 9.
Photomicrograph of lung tissues from LPS/zymosan-treated
(A, C), and ATK/LPS/zymosan-treated
(B, D) mice 4 h after LPS administration.
Hematoxylin-eosin stain. Scale bar in A represents 200 µm
in A and B and 50 µm in C and
D.
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DISCUSSION |
The results of the current study show that cPLA2 is
important in the pathogenesis of acute lung injury. Inhibition of
cPLA2 significantly attenuated acute lung injury induced by
endotoxemia. These observations indicate that pharmacological
inhibition of cPLA2 may be an effective treatment for acute
lung injury, probably because it inhibits production of inflammatory
mediators including TXs and LTs.
The sepsis syndrome is the most frequent cause of ARDS and is
associated with 35-45% incidence of ARDS development (9, 10). It is postulated that both endotoxemia and phagocytosis of
bacteria are involved in the pathogenesis of ARDS associated with
septic syndrome (6). Therefore, we used the current model of acute lung injury induced by combined administration of LPS and
zymosan (19). In this model, circulating LPS and
phagocytosis of bacterial particles by LPS-primed PMN elicit acute lung
injury, which may mimic sepsis-associated acute lung injury.
After LPS/zymosan administration, we observed increases in
EL, protein leakage, and PMN infiltration and severe
exacerbation of gas exchange. PMN infiltration in the lung was
confirmed by MPO activity assay and histology. Consistently, marked
increases in TXs and LTs were detected in the BALF. These findings were significantly attenuated by the treatment of cPLA2
inhibitor ATK. Potential mechanisms by which cPLA2 mediates
sepsis-induced acute lung injury include the release of proinflammatory
mediators. The present results also suggest that the major mediator of
PMN infiltration is a cPLA2 product, most probably
LTB4 (38). Recent evidence using lung injury
models overexpressing the LTB4 receptor shows that
LTB4 is an important mediator of neutrophil-mediated lung
injury (4). It is suggested that not only infiltration but
also activation of PMN in lungs may be essential to induce the
development of acute lung injury. The cPLA2-initiated
pathways may mediate both infiltration and activation of PMN triggered by septic syndrome, resulting in sepsis-associated ARDS. In human neutrophils during sepsis, elevated cPLA2 expression and
activity have been recently reported, suggesting that cPLA2
plays a major role in neutrophil function in septic syndrome
(17).
Of note, it has been recently shown that acute lung injury induced by
LPS/zymosan administration is attenuated in cPLA2
gene-disrupted mice (30). It seems that the effects of ATK
administration are similar to those of cPLA2 gene
disruption in terms of inhibiting lung injury. This observation may
further confirm that the intervention of cPLA2 could be an
effective approach to treat acute lung injury. However, differences
were also found between these two studies. In this study, we measured
TXB2, LTB4, and cysteinyl LTs
(LTC4/D4/E4) in BALF to confirm the
generation of cPLA2 products. Although the ATK
administration significantly attenuated LPS/zymosan-induced production
of TXB2, LTB4, and cysteinyl LTs, the ATK
administration reduced each eicosanoid by 73, 47, and 27%,
respectively, compared with LPS/zymosan administration. In contrast,
cPLA2 gene disruption reduced each eicosanoid by >90% in
this model, compared with LPS/zymosan administration in wild-type mice.
This finding suggests that the present manner of ATK administration may
still be insufficient to inhibit cPLA2 completely. Because
it is postulated that pharmacological intervention of cPLA2
could be useful in the management of ARDS, the development of novel
cPLA2 inhibitors warrants future research.
In the present model of acute lung injury, we observed that the
levels of PaCO2 and pH in the ATK/LPS/zymosan-treated
group were the same as in saline-treated controls. However,
LPS/zymosan-induced increases in EL, severity of hypoxia,
BALF protein, PMN, and eicosanoids were significantly attenuated but
not eliminated by the treatment of ATK. These observations indicate
that factors other than cPLA2 may also play a role and
contribute to physiological alteration. Recently, it has been
demonstrated that secretory PLA2 (sPLA2), the
other type of PLA2, mediates LPS-induced lung injury and
that the inhibition of sPLA2 may also represent a
therapeutic approach to acute lung injury (2). In
addition, it has been suggested that oxygen radicals, adhesion
molecules, and cytokines are also involved in this mechanism (8,
28). Recently, it was reported that cPLA2 activation
is essential for integrin-dependent adhesion of leukocytes
(39). If one considers that there are as yet no pharmacological agents to reverse pulmonary edema and increase survival
rates, these factors are potential targets to develop agents. The
current study suggests that the intervention of cPLA2 could
be a promising clue to improve management of ARDS.
In summary, the inhibition of cPLA2 significantly
attenuated lung damage and respiratory failure induced by LPS/zymosan
treatment. The current observations suggest that cPLA2
products are involved in the pathogenesis of acute lung injury caused
by septic syndrome. Inhibition of cPLA2-initiated pathways
might provide a novel and potential therapeutic approach to ARDS, to
which no pharmaceutical agents are currently available.
| |
ACKNOWLEDGEMENTS |
This work was supported in part by grants-in-aid for Scientific
Research from the Ministry of Education, Science, Sports and Culture of
Japan and grants-in-aid for Comprehensive Research on Aging and Health
from the Ministry of Health, Labour and Welfare, Japan, a grant from
the Mochida Memorial Foundation for Medical and Pharmaceutical
Research, a grant from the Yamanouchi Foundation for Research on
Metabolic Disorders, a grant from the Smoking Research Foundation, and
a grant from the Novartis Foundation for Gerontological Research. T. Aoki-Nagase is a Research Resident of Japan Foundation for Aging and Health.
| |
FOOTNOTES |
Address for reprint requests and other correspondence:
T. Nagase, Dept. of Geriatric Medicine, Faculty of Medicine,
Univ. of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan, 113-8655 (E-mail:takahide-tky{at}umin.ac.jp).
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. Section 1734 solely to indicate this fact.
First published December 27, 2002;10.1152/ajplung.00396.2002
Received 18 November 2002; accepted in final form 25 December 2002.
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