Yonago Acta medica 1998;41:45–52

The Relevance of Leukotriene B4 to the Development of Acute Lung Injury
Induced by Lipopolysaccharide
Kozo Hidaka and Yuichi Ishibe
Department of Anesthesiology and Reanimatology, Faculty of Medicine, Tottori University,
Yonago 683-0826, Japan
Acute lung injury (ALI) induced by lipopolysaccharide (LPS) develops by the activationof leukocytes via various mediators. Leukotriene B4 (LTB 4) has a strong effect on
activation and migration of leukocytes. We investigated the role of LTB 4 in the chain
leading to the development of ALI induced by LPS, by observing how an LTB 4 receptor
antagonist, ONO-4057, suppresses or mitigates leukocyte activation and migration. The
36 rabbits used in the experiment were divided into 3 groups: C group (control groupof 12 rabbits treated with physiological saline solution only); L group (of 12 rabbits
treated with 20 µg/kg LPS) and L-O group (of 12 rabbits treated with, first, 10 mg/kg
ONO-4057, then LPS). Blood samples were taken before, 3 h after and 6 h after the
injection of drugs; then the rabbits were exsanguinated. The right and left lungs were
removed for wet/dry weight ratio and bronchoalveolar lavage fluid (BALF)
measurements, respectively. We measured: the leukocyte counts in the peripheral blood,the chemiluminescence (CL) intensity to measure the amount of oxygen free radical
species (active oxygen species) production, the LTB 4 concentration in the blood, thecomplement activity levels (CH50), the polymorphonuclear neutrophil elastase (PMNE) and myeloperoxidase (MPO) levels in BALF, and the wet/dry weight ratio of theright lung. The leukocyte counts in L and L-O rabbits decreased significantly 3 h after
LPS injection, then were regained by the 6th h. Regarding CL (with and without
zymosan stimulation), there was no significant difference over time for C group. For L
group, the zymosan-stimulated CL showed a significant increase at the 6th h, whereas
the non-stimulated CL showed significant increases at the 3rd and 6th h. For L-Ogroup, the zymosan-stimulated CL showed a significant increase at the 6th h, whereas
the non-stimulated CL increased after 3 h, then slightly decreased after 6 h. The LTB4
levels showed significant increases at the 6th h for both L and L-O groups. The CH50
showed significant decreases at 6th h for both L and L-O groups. The MPO activity inthe BALF was significantly high for both the L and L-O groups. There was a tendency
for a high PMN-E level in the BALF for L group. The mean wet/dry weight ratio of theright lung was significantly high for L group, compared to both C and L-O groups.
Although an inhibitory effect on LTB 4 receptors by ONO-4057 failed to prevent
leukocyte migration, it successfully suppressed the activity of non-stimulated CL, MPO
and PMN-E, and, as a result, prevented the wet/dry weight ratio from increasing.
Key words: ALI; LPS; LTB4 ; LTB4 receptor antagonist

Acute lung injury (ALI) or acute respiratory
distress syndrome (ARDS) is caused, to a large
extent, by infections or endotoxemia by theendotoxin, lipopolysaccharide (LPS) (Knaus etal., 1994). Finding possible treatments for them isa subject of paramount clinical importance. LPS

stimulates macrophages, resulting in productionof humoral factors such as tumor necrosis factor(TNF), interleukin-1 (IL-1), interleukin-8 (IL-8),
leukotriene B4 (LTB 4 ) and so on (Said andHussein, 1989). These factors further stimulate
lymphocytic activity, resulting in the production45

K. Hidaka and Y. Ishibeof various mediators, which then form networksof their own by interacting with one another(Schlag and Redl, 1996). These mediator networks further aggravate inflammations, whichcause activation of neutrophils. Also, LPS directly activates alternative complement pathways(Said and Hussein, 1989). The neutrophils activated by LPS accumulate in the lungs (Pawe etal., 1982), then produce polymorphonuclear
neutrophil elastase (PMN-E) and oxygen free radical species via myeloperoxidase (MPO) activity(Idell et al., 1985; Said and Hussein, 1989).
Neutrophils normally act in defense of the bodybut, if activated excessively, migrate into the interstitum and alveoli and cause tissue damage, andultimately ALI. In order to prevent or minimize
the ill effects of neutrophils, therefore, the chainof neutrophil activation (described above) mustbe broken (Fisher et al., 1994). Among the mediators, the complement C5a, TNF-α, IL-1, IL-8,
platelet activating factor (PAF), LTB4, etc. areconsidered to be the major causes of ALI because of their strong effect on neutrophil activation and migration (Van Zee et al., 1991;
Vandermeer et al., 1995; Belcastro et al., 1996).
However, the role played by LTB4, which is ametabolic product of arachidonic acid and 5lipoxygenase, in LPS-induced ALI (Palmblad
et al., 1981; Yoshimura et al., 1994) has notbeen adequately studied so far. ONO-4057 wasdeveloped as an LTB4 receptor antagonist. In
vitro and in vivo, ONO-4057 has been shown tobe effective in inhibiting leukocyte migrationand degranulation; and in dermatitis experiments, it was shown to have an inhibitory effecton MPO activity (Kishikawa et al., 1992).
In this paper, we report on the results of ourexperiment designed to investigate the role
played by LTB 4 in the chain leading to thedevelopment of ALI triggered by LPS, by observing how an LTB4 receptor antagonist,
ONO-4057, suppresses or mitigates leukocyte
activation and migration.

taining to animal experiments, observed by the
Faculty of Medicine, Tottori University. We
used 36 rabbits (Japanese White rabbits; female; range of weight, 1.7-2.9 kg), which wererandomly divided into 3 groups: C group (control group of 12 rabbits to be treated with physiological saline solution only); L group (ofwhich 12 rabbits were to be injected with LPS)
and L-O group (of which 12 rabbits were to betreated with ONO-405 first, and then LPS). Initially, a sample of 7 mL of blood was takenfrom the ear vein of each rabbit in the 3 groups
before the administration of the drugs. The C
group rabbits, then, were given 12 mL of physiological saline solution. The L group rabbits
were given 7 mL of physiological saline solution and 5 mL of LPS solution (E.coli B8.,
Difco, Detroit, MI; LPS 20 µg per each kg of
rabbit weight, µg/kg, dissolved into physiological saline solution). The L-O group rabbits
were given, first, 5 mL of ONO-4057 (10 mg/kg) solution (20 mg/mL, dissolved into 7%
NaHCO 3, to which physiological saline solution was added to make the total to 5 mL); then,they were given 2 mL of physiological saline
solution for 2 min intravenously; and finally,
they were given 5 mL of LPS solution (20 µg/kg) through the ear vein. We let the rabbits freein the cages until the next blood sampling time.
Three h after the initial drug administration,
another sample of 2 mL of blood was takenfrom the ear vein; then the rabbits were given 2
mL of physiological saline solution intravenously.
We let them free again. Six hours after the LPS
injection, a sample of 7 mL of blood was takenfrom the ear vein. The rabbits, then, were given
20 mg/kg of pentobarbital intravenously, andwere anesthetized by 50 mg/kg of ketamine via
muscle injection. Then we initiated tracheotomy on the rabbits locally anesthetized with
1% lidocaine. Breathing was controlled by inserting a tube endotracheally and using a ventilator for small animals (Model 681, Harvard
Apparatus, South Natic, MA) set at 20 respirations/min, 6 mL of room air per kg body weight,
and 2 cm H2 O of positive end-expiratory pressure. The sternum (locally anesthetized with
1% lidocaine) was cut in the middle and, withthe thorax open, the pericardium was opened,

Materials and Methods
The animal experiment reported here was donein conformity with the ethical guidelines per46

Effect of LTB4 antagonist to ALI indueced by LPS

and the right ventricle was incised for exsanguination. The heart and the lungs (with the respiration tube inserted) were taken outside thebody as a unit. The right hilum was ligated, andthe right lung was removed for wet/dry weightmeasurement. Twenty milliliters of physiologicalsaline solution (5 mL over 4 times) were infused
into the left lung through the main trachea; thena sample of 12–15 mL of bronchoalveolar lavage fluid (BALF) was taken from the left lung.
The measurements were the leukocyte
counts in the peripheral blood; the chemiluminescence (CL)/leukocyte (n = 6 with zymosanstimulation and n = 6 without, for each group)
to measure the amount of oxygen free radical
species production; the LTB4 levels (n = 8 for C
group; n = 9 for L group and n = 9 for L-Ogroup); the complement activity levels (CH 50);
the PMN-E and MPO activity levels, from thesupernatants of the BALF, centrifuged at 1000
rpm, 10 min. The leukocytes were counted by
Celltac (MEK-5158, Nihon Kohden, Tokyo,
Japan). For CL, a Luminescence Reader (BLR201, ALOKA, Tokyo) was used to measure the
50-min luminescence of the 0.1 mL of heparinadded blood in 1.2 mL of luminol-HEPES buffer solution (14 mM HEPES, pH 7.4, containing

10

66 mM of luminol), stirred at 37˚C. Zymosanstimulated CL was measured when 0.1 mL of
40 mg/mL non-opsonized zymosan (zymosan
A: Sigma Chemical Co., St. Louis, MO) was
added; and non-zymosan-stimulated CL, without it. LTB4 was measured by the Radio Immuno
Assay method (Mitsubishi Chemical BCL,Tokyo). Complement activity was measured by
the CH50 method (SRL Inc., Tokyo). PMN-E
activity in the BALF (0.5 mL) was measured by
the hydrolysis method of Yoshimura and coworkers (1994): the hydrolysis of the substrate,
250 µM of L-hydroglutamyl-L-propyl-L-valin-pnitroanilide (Dai-ichi Chemicals, Tokyo), wasmeasured by a Multiskan MS (Labsystems,
Basingstroke, Helsinki, Finland) at 405 nm,
following the incubation with a Tris buffer (25mM, pH 8.3) at 37˚C for 4 h. MPO activity inthe BALF (0.5 mL) was measured by themethods described previously (Henson et al.,
1978; Belcastro et al., 1996): The absorption
density of o-dianisidine (96 µg/mL) was measured by the Multiskan MS at 480 nm (extinction coefficient: 1.13 × 10 4 /M), following
incubation with phosphorous buffer (38 mM,
pH 6.2) and 0.0038% H2O2 at 37˚C for 15 min.
The measured values are expressed as mean
and SEM. The statistical analysis was performed by an analysis of variance, followed by
Scheffé’s test (for differences among groups) anda paired Student’s t-test (for differences withingroups). A P value of less than 0.05 was considered statistically significant.

5

Results(× 103/mm3)

Leukocyte count

15

*

0

Before

*
**
**

3h

There was no statistically significant differencein the weights of the rabbits among all groups.
There was no significant difference overtime in leukocyte counts for C group, whereas
the leukocyte counts for L and L-O groups decreased significantly after 3 h, but they regained
after 6 h (Fig. 1).
Regarding CL (with and without zymosanstimulation), there was no significant difference

6hFig. 1. Changes in leukocyte counts for the 3 groups (n= 12, mean ± SEM, for each group). *Versus the previous counts of the same group (P < 0.05); **versus thecounts in the control group at each time (P < 0.05). ,C group (control group); , L group (20 µg/kg LPS); ,L-O group (20 µg/kg LPS and 10 mg/kg ONO-4057).

Abbreviations: ALI, acute lung injury; ARDS, acute respiratory distress syndrome; BALF, bronchoalveolar lavage
fluid; CH50, complement activity levels; CL, chemiluminescence; IL, interleukin; LPS, lipopolysaccharide; LTB4,
leukotriene B 4; MPO, myeloperoxidase; PAF, platelet activating factor; PMN-E, polymorphonuclear neutrophil
elastase; TNF, tumor necrosis factor

47

K. Hidaka and Y. Ishibe(kCounts)**(kCounts)

**
**

*

2.5

CL/leukocyte

CL/leukocyte

20

15

10

5

*

2.0
1.5

**

*
**
**

1.0
0.5

0

Before

3h

0.0

6h

Before3h6hWith zymosanWithout zymosan

Fig. 2. Changes in chemiluminescence (CL) intensity with zymosan stimulation for the 3 groups (n =6, mean ± SEM, for each group). *Versus theprevious intensity of the same group (P < 0.05);**versus the intensity of the control group at eachtime (P < 0.05). , C group (control group); , Lgroup (20 µg/kg LPS); , L-O group (20 µg/kg LPS
and 10 mg/kg ONO-4057).

Fig. 3. Changes in chemiluminescence (CL) intensity without zymosan stimulation for the 3 groups (n= 6, mean ± SEM, for each group). *Versus theprevious intensity of the same group (P < 0.05);**versus the intensity of the control group at eachtime (P < 0.05). , C group (control group); , Lgroup (20 µg/kg LPS); , L-O group (20 µg/kg LPS
and 10 mg/kg ONO-4057).

over time for C group. The zymosan-stimulated
CL for L group showed a significant increase
from 4.214 ± 1.397 kCounts/leukocyte count(before LPS injection) to 14.952 ± 0.965 (6 hafter), whereas the non-stimulated CL showed asignificant increase from 0.728 ± 0.210 (before
LPS injection) to 1.097 ± 0.320 (3 h after), thento 1.554 ± 0.467 (6 h after) (Figs. 2 and 3). Similarly, the zymosan-stimulated CL for L-Ogroup showed a significant increase from 4.556± 0.934 (before LPS infusion) to 15.648 ± 4.168(6 h after) (Fig. 2), whereas the non-stimulated
CL increased from 0.732 ± 0.234 (before LPS
injection) to 1.170 ± 0.245 (after 3 h), but it de-creased to 0.963 ± 0.235 (after 6 h) (Fig. 3).
The intergroup comparisons show that thezymosan-stimulated CL for L and L-O groups(6 h after LPS injection) was significantly
higher than that for C group, whereas the nonstimulated CL for L group (3 and 6 h after) was
significantly higher than that for C group, andthe non-stimulated CL for L-O group (3 h after)
was significantly higher than that for C group(Figs. 2 and 3).
While there was no significant difference in
LTB4 (before and 6 h after LPS injection)
among all groups, the LTB4 for L and L-Ogroups showed significant increases from

(pg/mL blood)
200(U/mL)

*

*

10

CH50

150

LTB 4

*

8

100

50

*

6
4
2
0

0

Before6h

BeforeFig. 4. Changes in leukotriene B4 (LTB4 ) concentration for the 3 groups (n = 12, mean ± SEM, foreach group). *Versus the previous concentration of
the same group (P < 0.05). , C group (control
group); , L group (20 µg/kg LPS); , L-O group(20 µg/kg LPS and 10 mg/kg ONO-4057).

6hFig. 5. Changes in complement activity for the 3groups (n = 12, mean ± SEM, for each group).
*Versus the previous activity of the same group (P <0.05). , C group (control group); , L group (20µg/kg LPS); , L-O group (20 µg/kg LPS and 10
mg/kg ONO-4057).

48

Effect of LTB4 antagonist to ALI indueced by LPS

12
10

**

(U/mL BALF)**

PMN-E activity

MPO activity(× 10–6 M/mL BALF)

8
6
4
2
0

10
8
6
4
2
0

Fig. 7. Polymorphonuclear neutrophil elastase(PMN-E) activity of bronchoalveolar lavage fluid(BALF) for the 3 groups (n = 12, mean ± SEM, foreach group). , C group (control group); , L group(20 µg/kg LPS); , L-O group (20 µg/kg LPS and 10
mg/kg ONO-4057).

121.12 ± 12.91 and 123.91 ± 12.88 pg/mL blood(before LPS injection) to 155.86 ± 19.89 and
166.89 ± 20.11 (6 h after), respectively (Fig. 4).
Regarding the CH50 for C group, there wasno significant difference between the time before physiological saline solution was given(6.5 ± 0.6 U/mL) and the time 6 h later (6.1 ±0.5), whereas for L and L-O groups, CH 50
showed significant decreases from 6.4 ± 0.4 and
6.4 ± 0.6 (before LPS injection) to 4.9 ± 0.6 and
5.0 ± 0.8 (6 h after), respectively (Fig. 5).
The MPO level in the BALF for C groupwas 1.77 ± 1.50 × 10 -6 M/mL BALF, whereas
those for L and L-O groups were significantly
higher at 8.54 ± 1.11 and 6.38 ± 1.03, respectively; and the MPO of L-O group tended to belower (P = 0.17) than that for L group (Fig. 6).
Although there was no significant difference in PMN-E in the BALF among groups, thePMN-E for L group tended to be higher (7.22 ±2.36 U/mL BALF) in contrast with those for C
group (6.85 ± 0.98, P = 0.26) and L-O group(6.76 ± 1.63, P = 0.25) (Fig. 7).

The mean wet/dry weight ratio for L groupwas significantly higher (5.13 ± 0.06) thanthose for C group (4.93 ± 0.06) and L-O group(4.98 ± 0.04) (Fig. 8).

Wet/dry ratio

Fig. 6. Myeloperoxidase (MPO) activity of bronchoalveolar lavage fluid (BALF) for the 3 groups (n= 12, mean ± SEM, for each group). **Versus the, C group (control
control group (P < 0.05).
group); , L group (20 µg/kg LPS); , L-O group(20 µg/kg LPS and 10 mg/kg ONO-4057).

5.5

**

Discussion
Our experiment showed that: i) ONO-4057 did
not block leukocyte migration caused by LPS;ii) while ONO-4057 did not have an effect on
the zymosan-stimulated CL of leukocytes (6 hafter LPS injection), it inhibited the non-stimulated CL; iii) there was a tendency for ONO4057 to inhibit PMN-E and MPO activities in
the BALF; and iv) ONO-4057 successfully inhibited the increase in wet/dry weight ratio.
The experiments using guinea pigs by
Kishikawa and coworkers (1992) showed that
ONO-4057 inhibited the LTB4-induced leukopenia in the peripheral blood in a dose-dependent manner, effective when 10 mg/kg or more
were ingested and when 0.3 mg/kg or more were
given intravenously. In our experiment, therabbits received intravenous injection of 10 mg/
kg of ONO-4057 2 min before LPS was injected
intravenously. The reason for our decision touse 10 mg/kg of ONO-4057 this time is because
there were 3 rabbits which were given 5 mg/kg
of ONO-4057, but which did not show any
effect of ONO-4057 to inhibit the LPS-induced
decrease in leukocyte count; and the rabbits
which were only given 10 mg/kg of ONO-4057
did not die nor did they show any signs ofabnormality such as weakness or a lowering of
their activity level. From these observations,

†

5.0

4.5

Fig. 8. Wet/dry ratios of the 3 groups (n = 12, mean
± SEM, for each group). **Versus the control group(P < 0.05). †Versus the L-O group (P < 0.05). , Cgroup (control group); , L group (20 µg/kg LPS);, L-O group (20 µg/kg LPS and 10 mg/kg ONO4057).

49

K. Hidaka and Y. Ishibe

we decided to try 10 mg/kg of ONO-4057, expecting sure signs of the effectiveness of ONO4057.
We decided on the dosage of LPS to be 20µg/kg, which was the dosage inferred from our
preparatory experiments, to be maximallyeffective without adversely affecting the designof our experiment. We also decided that ourobservation time should be 6 h, on the basis of
the significant results of other experiments inwhich observation time was 6 h, to measure theeffectiveness of certain drugs in inhibiting theactivation of neutrophil elastase (Kubo et al.,
1994; Nishina et al., 1997), and by taking into
consideration the time it takes for various kindsof cytokines to be produced (Sakamoto et al.,
1994; Salvemini et al., 1995; Nishina et al., 1997).
It has been reported that the concentrationlevel of LTB4 after LPS injection is inferred tohave 2 phases of change: the peak of LTB4 at
an earlier stage (1 h after) is induced by macrophages in the blood, whereas the LTB4 peak at alater stage (4 h after) is due to the leukocytes
accumulated in the lungs (Kobayashi et al.,1993). In our experiment, the LTB4 concentration levels for L and L-O groups showed increases at 6 h, thus not showing the effectiveness of ONO-4057 in inhibiting the productionof LPS-induced LTB4. We speculate that thiswas due to the fact that although ONO-4057
might have had an effect on LTB4 receptors,
neutrophils could still have produced LTB4 byother means (such as via complements, PAF, concentrated IgG, TNF, etc.) (Fogh et al., 1992).
It has been reported that with the rabbits
injected with LPS intravenously, there was anaccumulation of leukocytes in the lungs andliver (after 2 h) and decreases in the leukocyte
count in the peripheral blood, the bone marrow,and the spleen (Toft et al., 1994). From our preparatory experiments we knew that the injectionof LPS into rabbits decreased their leukocyte
count in the peripheral blood after 5 min; thisproved to be true for the L group in our present
experiment. Also, the leukocyte count even for
L-O group, like L group, significantly decreased 3 h after LPS injection, and ONO-4057 failed to inhibit leukocyte migration.
The amount of complement activity for L

and L-O groups significantly decreased 6 hafter LPS injection. We conjecture that this wasdue to the fact that the leukocytes accumulated
in the liver as well as in the lungs, and damaged
the liver; and the production of complements inthe liver could not catch up with the consumption of LPS-induced complements (Arai et al.,1989).
The non-stimulated CL intensity for L-Ogroup (as well as that for L group) showed ahigh value at 3 h; but it became lower after 6 h.
This, together with the fact that the LTB4 for LO group (as well as that for L group) was high at
6 h, indicates that there was a factor other than
LTB4 that contributed to the production of an
oxygen free radical species. LPS, directly andindirectly, stimulates and produces various
mediators and cytokines. It also stimulates C3
directly, and activates alternative complement
pathways (Said and Hussein, 1989). We thinkthat the higher non-stimulated CL intensity
levels of L and L-O groups, compared to thelevel of C group, at 3 h, were due to the fact thatcomplements were directly stimulated by LPS
and the neutrophil activity increased because ofother mediators and cytokines.
Zymosan activates complement components (after C3) via properdin-related factors.
The zymosan-stimulated CL intensity levels for
all groups before treatments and C group at 3
and 6 h were about 10 times higher than their
non-stimulated CL counterparts. This can be
explained by saying that the complements in the
blood reacted to zymosan.
The zymosan-stimulated CL intensity levels
for L and L-O groups were significantly high at
6 h, and the non-stimulated CL level of L-Ogroup decreased after 6 h. This can be explained in terms of the Second Attack Theory(Ogawa, 1996). According to the theory, we
can say that the LPS in L and L-O groups
heightened the responsiveness of the neutrophils, which zymosan further stimulated as asecond attack, resulting in more production ofoxygen free radical species and, consequently,
an increase in CL. The amount of oxygen free
radical species production in the peripheral bloodin L-O group, when no additional zymosaninduced stimulation was present, can be known
50

Effect of LTB4 antagonist to ALI indueced by LPS

from the amount of oxygen free radical species
production without zymosan stimulation at 6 h.
We can attribute to the effectiveness of ONO4057 the fact that the production capacity ofoxygen free radical species from leukocytes in
the peripheral blood is lower for L-O group thanfor L group. From these facts, we inferred that
ONO-4057 had an inhibitory effect on LTB 4
receptor activity and, thus, helped lower the
production of oxygen free radical species fromneutrophils; but ONO-4057 failed to control the
LPS-heightend responsiveness of neutrophils.
The levels of the MPO and PMN-E for L-Ogroup tended to be lower than those for L group,
indicating a weak positive effect of ONO-4057.
The amount of oxygen free radical species increases and decreases, depending on whether
PMN-E is present or not, respectively (Kusner andKing, 1989). Also, one of the PMN-E inhibitors,
α1-protease inhibitor (α1-PI), becomes inactivated in the presence of oxygen free radical
species (Tanaka et al., 1991). Thus, oxygen free
radical species and PMN-E influence each other.
The wet/dry weight ratio indicates an increase invascular permeability (Suzuki et al., 1994) due tothe damage done to endothelial cells (Tate andRepine, 1983) jointly by oxygen free radical
species and PMN-E. The results of our experiment indicate that ONO-4057 must have had an
inhibitory effect (up to 6 h) on the LPS-induced
increase in vascular permeability in the lungs.
From the above discussion, we can say that
ONO-4057 was effective against lung injuriescaused by LPS, but with a few limitations.
First, the intravenous injection of ONO-4057
caused our rabbits to show signs of pain, anddeath in some cases with LPS after ONO-4057
injection. We used ONO-4057 diluted in physiological saline solution and injected it slowly,
but there is a possibility that the pain caused by
ONO-4057 injection might have led to the production of various mediators, cytokines, etc.
Also, while LPS 20 µg/kg alone did not kill any
rabbits, pretreatment with ONO-4057 killed 3
out of 15 rabbits within 1.5 h to 2 h. However,
the rabbits given LPS alone were weak, suffering from diarrhea, whereas the rabbits whichwere pretreated with ONO-4057 and which survived 6 h until the end of the experiment, exhibited no sign of diarrhea or any abnormality. We
know that the lethal dose of ONO-4057 given
intravenously for rats is 300 mg/kg*; but welack similar data pertaining to rabbits. It might
be that for rabbits, the 50% effective dose of
ONO-4057 is very close to the 50% lethal dose.
Also, this might indicate the complexity of
mediator networks. We speculate that in ourdead rabbit cases, the antagonistic effect of
ONO-4057 was so strong on LTB4 receptors
that LTB4, which is necessary for normal physiological functioning, was inhibited, and consequently, parts of the mediator networks were
blocked, which caused a malfunction in homeostasis in those dead rabbits.
Conclusions
Although ONO-4057, as an LTB4 receptor antagonist, did not prevent leukocyte migration
caused by LPS, it tended to control the nonstimulated CL level of leukocytes in the peripheral blood, and suppressed the MPO and PMNE activity in the BALF, as a result, preventing
the wet/dry ratio from increasing. From theseresults, we conclude that LTB4 must be playing
a role in LPS-caused lung injuries. However,
our experiment also suggests the possibility thatan inhibitory effect on LTB4 receptors might
adversely lead to the suppression of the naturalprotective mechanism by LTB4 against inflammation, and might further lead to the breakdown of mediator networks.
Acknowledgments: The authors thank Drs. N.
Okazaki and R. Liu (Dept. of Anesthesiology andReanimatology) and Dr. N. Nakada (Third Dept. ofInternal Medicine), Faculty of Medicine, Tottori
University for their help and advice, and Dr. H.
Nagahara, Dept. of East Asian Languages andLiteratures, University of Hawaii, for his help with
the English. The authors also thank Ono Pharmaceutical Co., Osaka, Japan, for their generous supplyof ONO-4057.
References*Ono Pharmaceutical Co., Tokyo; unpublished research report.

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(Received December 22, 1997, Accepted January 27, 1998)

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