NAOSITE: Nagasaki University's Academic Output SITENAOSITE: Nagasaki University's Academic Output SITE
In vivo efficacy of sivelestat in combination with pazufloxacin
against Legionella pneumonia.
Morinaga, Yoshitomo; Yanagihara, Katsunori; Araki, Nobuko;
Yamada, Koichi; Seki, Masafumi; Izumikawa, Koichi; Kakeya,
Hiroshi; Yamamoto, Yoshihiro; Yamada, Yasuaki; Kohno,
Shigeru; Kamihira, Shimeru
CitationExperimental lung research, 36(8), pp.484-490; 2010
© 2010 Informa Healthcare USA, Inc. / This is an electronic version of an
article published in Experimental lung research, 36(8), pp.484-490; 2010.
Experimental lung research is available online at:
Y. Morinaga et. al. 1 1 / 1919
In Vivo Efficacy of Sivelestat in Combination with Pazufloxacin
against Legionella Pneumonia
Yoshitomo Morinaga1,2, Katsunori Yanagihara1,2, Nobuko Araki2, Koichi Yamada1,2, Shigeki
Nakamura2, Masafumi Seki2, Koichi Izumikawa2, Hiroshi Kakeya2, Yoshihiro Yamamoto2,
Yasuaki Yamada1, Shigeru Kohno2,3 and Shimeru Kamihira1
1Department of Laboratory Medicine, Nagasaki University Graduate School of Biomedical
Sciences, Nagasaki, Japan
2Second Department of Internal Medicine, Nagasaki University Graduate School of
Biomedical Sciences, Nagasaki, Japan
3Global COE Program, Nagasaki University, Nagasaki, Japan
Running title: Effects of Sivelestat on Legionella Pneumonia
Address correspondence to:
Katsunori Yanagihara, MD, PhD
Department of Laboratory Medicine
Nagasaki University Graduate School of Biomedical Sciences
1-7-1 Sakamoto, Nagasaki 852-8501, Japan
Tel: +81-95-819-7418; Fax: +81-95-819-7257
Y. Morinaga et. al. 2 2 / 1919
It is important to regulate excessive inflammation when we treat patients with severe
infectious disease. Sivelestat sodium hydrate (sivelestat), a neutrophil elastase inhibitor, is
used in the treatment of lung injury but its effect on pneumonia is unknown. We examined the
efficacy of sivelestat in combination with a fluoroquinolone, in a Legionella pneumophila
pneumonia mouse model. The combination therapy did not show a significant survival
improvement compared to the treatment with fluoroquinolone alone, but reduced bacteria
number and inflammatory cells in the early phase. The combination therapy can contribute to
treatment of L. pneumophila pneumonia with protecting lungs.
Neutrophil elastase, Legionella pneumophila, severe pneumonia
Y. Morinaga et. al. 3 3 / 1919
Neutrophils play important roles in the host’s defense against pathogens by collaborating
with other immune cells. The infiltration of activated neutrophils into the lungs and their
migration into the airways are major features in response to pulmonary infection and
inflammation . These activated neutrophils produce a variety of pro-inflammatory
cytokines, such as interleukin-8 (IL-8), interleukin-1 (IL-1) and tumor necrosis factor-
(TNF-) , and secrete numerous proteases, in particular neutrophil elastase (NE) .
Neutrophil elastase is a nonspecific serine protease that has bactericidal and
pro-inflammatory activities [4-5], but is also cytotoxic and can cause tissue damage under
conditions that induce high expression, including acute lung injury (ALI) and acute
respiratory distress syndrome (ARDS) . Neutrophil elastase also plays a pathogenic role in
pulmonary emphysema and chronic inflammatory airway diseases [7-8].
The specific NE inhibitor, sivelestat sodium hydrate (sivelestat), has been shown to decrease
neutrophil counts in bronchoalveolar lavage fluid (BALF) and lung damage in acute lung
injury animal models [9-10]. In Japan, sivelestat is used in the treatment of ALI and ARDS
patients with systemic inflammatory response syndrome and its clinical effectiveness has
been documented [11-12].
Legionella pneumophila, which is a Gram-negative intracellular bacterium, causes serious
pneumonia in humans. L. pneumophila pneumonia often progresses rapidly with severe
systematic inflammation and is sometimes life-threatening , despite the strong activity
fluoroquinolone and macrolide antibiotics have against Legionella species . A variety of
host cells, including macrophages and neutrophils, are involved in the immune response to L.
pneumophila [15-16]. However, the accumulation of activated neutrophils can lead to the
Y. Morinaga et. al. 4 4 / 19 19
production of excessive NE, which causes lung tissue damage. Thus excessive inflammation
may play a role in part in the pathogenesis of severe pneumonia. We previously reported the
effectiveness of sivelestat in a mouse model of severe pneumococcal pneumonia  and its
effective case with Legionnaire’s disease was reported , however, its efficacy against
severe pneumonia is still unclear.
Pazufloxacin is a parenteral fluoroquinolone antibiotic available in Japan that has potent
activity against Legionella species . However, treating patients with severe pneumonia
using antibiotics alone is difficult and thus additional therapies aimed at the various aspects
of Legionella pathology are required. To confirm whether sivelestat improves the acute
inflammation and/or survival, we examined the efficacy of sivelestat in combination with the
fluoroquinolone pazufloxacin, in a murine model of severe L. pneumophila pneumonia.
Materials and methods
Sivelestat was kindly provided by Ono Pharmaceutical Co., Ltd. (Osaka, Japan).
Pazufloxacin was kindly provided by Taisho Toyama Pharmaceutical Co., Ltd. (Tokyo,
Japan). Both reagents were dissolved in physiological saline just before use.
Male 8-week-old A/J mice were purchased from Japan SLC, Inc. (Sizuoka, Japan). All
animals were housed in a pathogen-free environment and received sterile food and water in
the Laboratory Animal Center for Biomedical Science at Nagasaki University. Animal care
and experimental procedures were performed in accordance with the Guidelines for Animal
Y. Morinaga et. al. 5 5 / 19 19
Experimentation of Nagasaki University with approval of the Institutional Animal Care and
In this study we used the L. pneumophila NUL1 strain, which was clinically isolated from
the sputum of a patient at Nagasaki University hospital. The bacteria were stored at -80°C in
a Microbank system (Pro-Lab Diagnostics, Ontario, Canada) until use.
Antibiotic susceptibility testing
The MIC of pazufloxacin and sivelestat against NUL1 was determined using the
microdilution method with ACES-buffered yeast extract supplemented with -ketoglutarate
broth [(19)]. Microtiter plates containing 5.0 × 105 cfu/well were incubated with pazufloxacin
or sivelestat at 37°C for 72 h, and the lowest concentration of the drug that prevented visible
growth was considered the MIC.
Experimental model of Legionella pneumophila pneumonia
To prepare the inoculum, NUL1 was cultured on a buffered charcoal yeast extract
(BCYE) agar plate for 72 h, then the bacteria suspended in saline, harvested by
centrifugation (3,000 g, 4°C, 10 min), resuspended in sterile saline and adjusted
approximately to 2 × 109 cfu/ml, as estimated by turbidimetry. Anaesthetized mice were
inoculated with the bacteria at 50 l/mouse (1 × 108 cfu/mouse) intratracheally .
Treatment commenced 24 h after inoculation. Sivelestat alone (4.8 mg/kg; SIV mice),
Y. Morinaga et. al. 6 6 / 19 19
pazufloxacin alone (5.0 mg/kg; PZFX mice), or a combination of sivelestat and pazufloxacin
(the same dose of each drug as used alone; SIV+PZFX mice) were injected intraperitoneally
into the mice twice a day. In the control group (CTRL), saline was injected into the mice
instead of sivelestat or pazufloxacin. Viable bacteria counts and cytokines in the lungs, cell
counts in BALF and histopathological examinations were analyzed in each group on day 2
(12 h after the 2nd treatment). The survival rates of mice were analyzed after 7-day treatment.
The mice were sacrificed by cervical dislocation on day 2 (12 h after the 2nd treatment). The
lungs were dissected under aseptic conditions and suspended in 1 ml saline. The organs were
homogenized with a homogenizer (AS One Co., Osaka, Japan), quantitatively inoculated onto
BCYE agar plates using serial dilutions and incubated at 37°C for 72 h to count viable
The mice were sacrificed by cervical dislocation on day 2. Lung tissue was fixed in 10%
buffered formalin and stained with hematoxylin-eosin.
Concentrations of MIP-2, TNF- and IL-1 in the lung were assayed using mouse cytokine
enzyme-linked immunosorbent assay (ELISA) test kits (R & D Systems, Minneapolis, MN,
USA) according to the manufacturer’s instructions.
Y. Morinaga et. al. 7 7 / 1919
Bronchoalveolar lavage (BAL) fluid examination
BAL was performed as described previously . Briefly, mice were sacrificed on day 2
after inoculation. The chest was opened to expose the lungs and a disposable sterile plastic
cut-down intravenous catheter was inserted into the trachea. BAL was performed three times
sequentially using 1 ml saline each time and the recovered fluid fractions were pooled for
each animal. Fluid recovery was routinely 90%. Leukocytes in BALF samples obtained
from each mouse were washed and counted with a hemocytometer. For differential cell
counts, cells were centrifuged at 1,000 × g for 1 min and then fixed onto slides, which were
then stained with Diff-Quik stain. Differential cell counts were performed by counting 100
Data represented the mean ± standard error of the mean (SEM) and statistical analyses were
performed using Scheffe’s test following the Kruskal-Wallis non-parametric test. Statistical
significance was defined as p < 0.05. Survival analysis was performed by Kaplan-Maier
The MICs of pazufloxacin and sivelestat against the NUL1 strain were 0.063 and >128
Y. Morinaga et. al. 8 8 / 19 19
The number of viable bacteria in the lungs was analyzed on day 2. The mean number of
viable bacteria in the lungs of CTRL, SIV, PZFX, and SIV+PZFX mice was 6.49 ± 0.19, 6.01
± 0.24, 5.93 ± 0.37 and 4.96 ± 0.44 log10 cfu/lung (mean ± SEM, n = 4 to 6), respectively
(Fig. 1). There were no sterile mice after treatment in any group. The number of viable
bacteria in the lungs of the SIV+PZFX mice was significantly fewer than that in lungs of the
CTRL mice (p = 0.045).
Cytokines in the lung
The concentrations of MIP-2 (Fig. 2a), IL-1 (Fig. 2b) and TNF- (Fig. 2c) in the lungs
were analyzed on day 2. Although the cytokine levels of the PZFX and SIV+PZFX mice
appeared to be less than those of the CTRL mice, there were no significant differences.
The total cell counts in BALF on day 2 of the CTRL, SIV, PZFX, and SIV+PZFX mice
were 2.78 ± 0.45, 2.02 ± 0.37, 2.32 ± 0.29 and 1.01 ± 0.25 105 cells/mL (mean ± SEM, n =
2 to 6), respectively (Fig. 3a). The percentage of neutrophils in BALF of the CTRL, SIV,
PZFX and SIV+PZFX mice were 79.5 ± 0.5, 75.8 ± 5.8, 88.0 ± 0.6 and 58.0 ± 8.5% (mean ±
SEM, n = 2 to 6), respectively (Fig. 3b). There was a statistical difference between the
percentage of neutrophils in BALF between PZFX and SIV+PZFX mice (p = 0.039).
The histopathological examinations of the lung specimens from mice sacrificed on day 2 are
shown in Fig. 4. In the CTRL mice, numerous neutrophils infiltrated into the alveolar spaces
Y. Morinaga et. al. 9 9 / 1919
and some alveoli were occupied by neutrophils (Fig. 4a). In the SIV (Fig 4b) and the PZFX
(Fig 4c) mice, many neutrophils were also observed but slightly fewer than in the CTRL mice.
Furthermore, in the SIV+PZFX mice we found noticeably fewer inflammatory cells
compared with the CTRL mice (Fig. 4d).
The survival rates of each group were analyzed after a 7-day-treatment (Fig. 5). All CTRL
mice had died by day 6, but over 60% of mice survived at least until day 8 in the other groups.
The SIV+PZFX mice demonstrated the best survival, with a significant difference compared
to CTRL mice (p = 0.010, Kaplan-Meier).
It is important for the treatment of severe infectious diseases to regulate the excessive
inflammation. We studied the efficacy of the combination therapy of sivelestat and a
fluoroquinolone antibiotic in a mouse model with severe L. pneumophila pneumonia. This
study demonstrated that the combination therapy can contribute to treatment of L.
pneumophila pneumonia with attenuating of lung inflammation derived from excessive NE in
the acute phase.
L. pneumophila is a pathogen that causes pneumonia with rapid worsening and is sometimes
life threatening. L. pneumophila infects macrophages initially and the infected macrophages
produce a variety of mediators, which subsequently stimulate inflammatory cells, including
neutrophils, T cells, natural killer cells and B cells . A previous study demonstrated that
NE levels are elevated and the mean anti-elastase capacity decreased in pneumonic lobes of
Y. Morinaga et. al. 1010 / 19 19
patients with community-acquired pneumonia . Although activated neutrophils are
considered to play a role in the host’s defense against invading microbial pathogens,
paradoxically they can produce excessive NE that damages lung tissue in severe pneumonia.
L. pneumophila is very susceptible to several antibiotics, such as the fluoroquinolones,
ketolides and macrolides . Pazufloxacin, a fluoroquinolone antibiotic available in Japan,
is also effective against Legionella [19, 23]. In this study, pazufloxacin showed slight
decrease of the number of viable bacteria, however, the combination therapy showed more
effective for decreasing of bacteria with a significant difference in spite of the short duration
of treatment. This suggests that the sivelestat supports the host’s defense against Legionella,
because sivelestat does not have antibacterial activity against NUL1.
MIP-2, IL-1 and TNF- are produced mainly by macrophages in L. pneumophila infection
 and mediate neutrophil recruitment and migration into lung tissue and alveolar spaces. In
particular, TNF- also enhances the bactericidal activity of macrophages and neutrophils and
stimulates the production of NE from neutrophils . Furthermore, these cytokines are also
produced by activated neutrophils . In results of cytokine concentration, we could not
observe the effectiveness of sivelestat and/or pazufloxacin. These results indicate that the
production of these cytokines is independent of sivelestat at least in the acute phase.
The marked number of neutrophils in BALF is a major finding in patients with Legionella
pneumonia . As shown in Fig. 3b, Legionella pneumonia mouse model also showed an
elevated percentage of neutrophils. The percentage of neutrophils was not suppressed in the
PZFX mice but slightly reduced in the SIV+PZFX mice. This finding implies that the
migration of neutrophils into the airway lumen is induced not only by bacteria but also by
active NE. The paradoxical results between leukocyte chemoattractant cytokines and cell
fraction in BALF may derive from the time lag. In addition, the histopathological findings
Y. Morinaga et. al. 11 11 / 19 19
agreed well with BALF examination. The findings in the SIV+PZFX mice seemed to be the
most effectively improved. Because NE can degrade proteoglycans in the glycocalyx and
components of the endothelial basement membrane , the combination therapy may
inhibiting the vicious cycle. Thus, NE can partly play a role in the pathogenesis in L.
The prolonged survival of the SIV+PZFX mice were most likely due to the improved
bacteriological and inflammatory findings in the SIV+PZFX mice after 2 treatments but there
was no significant difference compared with PZFX mice. Surprisingly, SIV mice also
survived in this study. Although we cannot clearly explain the reason of this survival
improvement, the reduced inflammatory cells in histopathological findings may be a sign of
this effect. We need further analyses about this.
The continuous infusion of sivelestat has previously been shown to decrease NE activity and
lung hemorrhage in lung injury animal models induced by acid- or endotoxin-inhalation [9,
25]. Thus, the lower effectiveness in this study may be due to the difference in drug
Finally, the A/J mouse used in this study is known as a susceptible animal to Legionella
because of Naip5 gene mutation concerning permissiveness to intracellular Legionella
replication, whereas other mice are resistant to Legionella . Therefore, alternative
approaches may be needed to explain the effectiveness of the combination therapy of
sivelestat and antibiotics against L. pneumophila pneumonia.
In conclusion, the combination therapy of sivelestat and pazufloxacin showed similar
survival rate of the treatment with pazufloxacin alone, but decreased the number of viable
bacteria in the acute phase in a mouse model of L. pneumophila pneumonia. We found a
significant difference in the percentage of neutrophils in BALF between the combination
Y. Morinaga et. al. 1212 / 1919
therapy and the treatment with pazufloxacin alone. These findings imply that sivelestat can
participate in the regulation of infection and inflammation. Treatment with sivelestat in
addition to antibiotics can be expected to be effective against L. pneumophila pneumonia
with reducing lung damage due to activated NE.
The first two authors (Y. M.and K.Y.) contributed equally to this work.
Y. Morinaga et. al. 13 13 / 1919
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