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Antimicrobial Agents and Chemotherapy, September 2001, p. 2638-2642, Vol. 45, No. 9
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.9.2638-2642.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Pretreatment of Mice with Clindamycin Improves
Survival of Endotoxic Shock by Modulating the Release of
Inflammatory Cytokines
Norio
Hirata,
Kazufumi
Hiramatsu,
Kenji
Kishi,
Tohru
Yamasaki,
Tomoku
Ichimiya, and
Masaru
Nasu*
Second Department of Internal Medicine, Oita
Medical University, Hasama, Oita 879-5593, Japan
Received 27 December 2000/Returned for modification 2 March
2001/Accepted 31 May 2001
 |
ABSTRACT |
Suppression of endotoxin release and subsequent production of
inflammatory cytokines is crucial in the treatment of septic shock. We
investigated the effect of clindamycin (CLI) on endotoxic shock induced
in mice by Escherichia coli lipopolysaccharide (LPS). Mice
were treated with CLI (160 to 600 mg/kg) or saline and then injected
with E. coli LPS and D-(+)-galactosamine
intraperitoneally 0.5 h after CLI administration. Pretreatment with CLI
significantly improved survival in a dose-dependent manner (CLI, at
160, 300, and 440 mg/kg) and significantly lowered the peak
concentrations of tumor necrosis factor alpha and
interleukin-1
(IL-1) in serum. However,
the peak concentrations of IL-6 in the sera of CLI-treated mice were
higher than in control mice. Our findings suggest that CLI alters
LPS-induced inflammatory cytokine production and suppresses endotoxin-induced mortality in this murine model.
| |
TEXT |
Septicemia is frequently associated
with serious complications, such as disseminated intravascular
coagulation and multiple organ failure, and the mortality of patients
with septicemia remains high despite recent advances in treatment with
antibacterial agents. It has been reported that about 400,000 patients
per year suffer from septicemia in the United States and that about
25% of these patients ultimately die of septic shock. Of patients with
positive blood cultures, about half showed growth of gram-negative
bacilli (11).
Septic shock induced by gram-negative bacilli has been reported to
result from overproduction or excessive release of inflammatory cytokines (e.g., tumor necrosis factor [TNF] and interleukin-1 [IL-1]) from immunocytes such as monocytes and macrophages, i.e., cells immunologically activated by endotoxin, which is a structural component (lipopolysaccharide [LPS]) of the cell wall of
gram-negative bacilli (20). Shock is induced through a
complex cytokine cascade triggered by these cytokines (3,
11). Many attempts have been made to suppress the actions of
these inflammatory cytokines (8, 14). However, since the
development of septic shock is due to complex interactions, no adequate
results have yet been obtained in clinical practice.
Studies have also been conducted using antibacterial agents which have
an anticytokine action (1, 10). Quinolones and macrolides
have been reported to suppress the production of IL-1 or TNF from
LPS-stimulated human monocytes (5, 12). Furthermore, the
administration of fosfomycin (FOF) led to a reduction in serum TNF
alpha (TNF-
) and IL-1 concentrations in LPS-stimulated mice (9). Clindamycin (CLI), which is effective against
gram-positive and anaerobic bacteria, has been demonstrated to induce
neutrophil phagocytosis in the host at subMIC levels (18,
19). We have previously reported that CLI significantly
suppressed TNF- release from purified LPS-stimulated human acute
monocytic leukemic cell line (THP-1) in vitro (7). In the
present study, we extended these early findings by conducting a study
in mice, on the assumption that CLI would also suppress inflammatory
cytokine release in vivo.
Effect of CLI administration time on survival of mice with
endotoxic shock.
An endotoxic shock mouse model using
D-(+)-galactosamine (GalN) and purified LPS (derived from
Escherichia coli O55:B5; Sigma Chemical Co. St. Louis, Mo.)
was used according to the modified method of Galanos et al.
(4). Briefly, GalN and purified LPS were diluted with
sterilized physiological saline and then 720 mg of GalN solution and 40 µg of purified LPS solution per kg were each administered
intraperitoneally to fasted, 10-week-old, specific-pathogen-free, male
C3H/HeN mice (Charles River Japan, Inc., Kanagawa, Japan)
(6). The survival rates of mice treated intraperitoneally
with 440 mg of CLI (Pharmacia & Upjohn, Tokyo, Japan), which is
commercially available as Dalacin S Injectable, per kg at various times
before and after the administration of GalN and LPS are shown in Table
1. Mice were observed for up to 3 weeks
after GalN and LPS administration to document the survival rate. All
saline-treated control mice died within 12 h of GalN and LPS
injection. In contrast, none of the mice treated with CLI at 0.5 h
before administration of GalN and LPS died. Furthermore, the survival
rates of mice treated with CLI at 2 or 6 h before administration
of GalN and LPS were significantly improved (78.6 and 85.7%,
respectively) compared to the control group (P < 0.01). On the other hand, the survival rates of the groups treated
with CLI at 0.5 and 1 h after administration of GalN and LPS were
significantly decreased (28.6 and 21.4%, respectively; P < 0.01). All mice in the group treated with CLI at 2 h after
administration of GalN and LPS died.
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TABLE 1.
Survival rate of mice treated with CLI (440 mg/kg) before
and after GalN+LPS administration in the endotoxin shock murine
model
|
|
Effect of CLI dosage on survival of mice with endotoxic shock.
The survival rates of fasted mice treated with CLI (160, 300, 440, 520, or 600 mg/kg) and the control group injected with GalN and LPS at
0.5 h after administration of CLI or saline are shown in Fig.
1. Mice in the control group began to die
about 6 h after administration of GalN and LPS, and all mice were dead within 12 h. In contrast, the survival rates in the CLI pretreated groups tended to improve in a dose-dependent fashion, with 100% survival in the group pretreated with a 440-mg/kg dose of CLI. However,
survival rates in groups treated with 520- and 600-mg/kg doses of CLI
decreased to 92 and 36%, respectively.
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FIG. 1.
Effect of CLI on survival rate after LPS and GalN
administration. Mice were treated with 160 ( , n = 14), 300 ( , n = 14), 440 ( , n = 14), 520 ( , n = 12), or 600 ( , n = 11) mg of CLI per kg or with saline alone ( , n = 21) at 0.5 h before GalN and LPS challenge. Survival rates were
scored at 48 h after the GalN+ LPS challenge, and the statistical
significance was determined by the Fisher's exact test (*,
P < 0.01 versus the control). No further mortality was
observed between day 2 and day 21 of followup.
|
|
Effects of CLI pretreatment on cytokine concentrations in
serum.
To elucidate the mechanism of improved survival following
CLI treatment, we determined the concentrations of cytokines over time
in serum. Mice in the CLI-treated groups (CLI at 160, 300, and 440 mg/kg) and the saline-treated control group were used for serum
sampling. GalN and LPS solutions were administered intraperitoneally to
the fasted mice at 0.5 h after pretreatment with CLI or saline. Mice were then sacrificed at 0.5, 1, 2, 4, and 6 h, and cardiac blood samples were immediately obtained. The blood sample was immediately centrifuged, and serum was stored at 
20°C until
measurement of cytokine concentrations. Concentrations of TNF-,
IL-1, and IL-6 in serum were determined by using an enzme-linked
immunosorbent assay (ELISA) kit (Cytoscreen; BioSource International,
Camarillo, Calif.). Concentrations were determined for two wells in
each sample. The TNF- concentration in serum began to increase
1 h after the administration of GalN and LPS, reached a peak at
2 h (1,183 ± 578 pg/ml), and declined thereafter in the
saline-pretreated control group (Fig.
2A).
TNF- concentrations in serum
decreased in a dose-dependent manner in all groups pretreated with CLI. Treatment with CLI (300 or 440 mg/kg) reduced TNF- concentrations in
serum to 562 ± 281 pg/ml (P < 0.01 versus the
control) and 339 ± 197 pg/ml (P < 0.01 versus
the control) at 2 h after administration of GalN and LPS,
respectively. These concentrations were significantly lower than those
in the control group. In the control group, IL-1 concentrations in
serum increased with time following administration of GalN and LPS and
reached a peak (228 ± 200 pg/ml) 6 h after administration
(Fig. 2B). The IL-1 concentrations in serum at 6 h after the
administration of GalN and LPS in mice treated with 300 and 440 mg of
CLI per kg were 31 ± 37 and 62 ± 63 pg/ml, respectively, significantly lower than in the control group (P < 0.01 and P < 0.05, respectively). The IL-6
concentration in serum began to rise from 0.5 h after administration of
GalN and LPS and reached a peak at 2 h in the control group (Fig.
2C). IL-6 concentrations in serum for groups treated with CLI at 160 and 300 mg/kg reached peak levels at 29.6 ± 10.0 and 37.4 ± 9.8 ng/ml, respectively, at 2 h after the administration of GalN
and LPS. These levels were significantly higher than in the control
group (P < 0.01 for both groups). The IL-6
concentration in serum in mice treated with a 440-mg/kg dose of CLI
reached a peak (26.2 ± 28.9 ng/ml) at 4 h after
administration of GalN and LPS, a value significantly higher than that
noted in the control group (P < 0.01).
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FIG. 2.
Effect of CLI on serum concentration of TNF- (A),
IL-1 (B), or IL-6 (C) in mice. Mice were treated with 160 ( ), 300 ( ), or
440 () mg of CLI per kg or with saline alone () at 0.5 h
before GalN and LPS challenge. TNF-, IL-1, and IL-6
concentrations in serum were determined by ELISA at 0.5, 1, 2, 4, and
6 h after GalN and LPS challenge. Each bar represents the
mean ± the standard deviation of 12 mice. Significant differences
in cytokines concentrations between groups were assessed by using
Student's t test (*, P < 0.01; **,
P < 0.05 versus the control).
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|
In the present study, pretreatment with an intraperitoneal injection of
440 mg of CLI per kg at 0.5 h before challenge with
GalN and LPS
resulted in an improvement of survival rate to 100%.
However,
administration of CLI after GalN and LPS challenge resulted
in only
partial improvement of survival. These findings suggest
that it took
about 0.5 h to exert CLI effect on cells that release
cytokines,
such as monocytes. At doses of CLI higher than 440
mg/kg, survival
rates tended to be lower. Since the 50% lethal
dose of CLI following
intraperitoneal administration in mice has
been reported to be 997 mg/kg (
17), CLI toxicity was suspected
to be responsible
for this
finding.
The results of TNF-
were in concordance with the direct effect of
CLI on THP-1 cells that was observed in our previous in
vitro study
(
7) and also with a report by Stevens et al.
(
15)
that CLI suppressed the release of TNF-
from
LPS-stimulated human
peripheral blood mononuclear cells. It has been
assumed that when
elevated by LPS stimulation, inflammatory cytokines
such as TNF-
and IL-1
serve as mediators of endotoxic death
(
3,
11).
In the endotoxic shock model used in the present
study, CLI-mediated
reductions in TNF-
and IL-1
concentrations
were considered to
contribute to the improved survival of
mice.
IL-6, a pleiotropic cytokine involved in immunological reaction
regulation and hemopoiesis in the acute phase, has been demonstrated
to
suppress LPS-stimulated production of TNF-
and IL-1
in cultured
human monocytes and in mice (
13,
16). Barton et al.
(
2)
ascertained that IL-6 increased the survival rate of
mice in an
LPS-GalN endotoxic shock mouse model. In the present study,
the
administration of CLI resulted in a rise in IL-6 concentrations
and
a fall in TNF-
and IL-1
concentrations in serum. We speculate
that the elevated IL-6 concentration in serum suppressed inflammatory
cytokine release, thereby contributing to improved survival rates,
although no detailed mechanism of CLI action on IL-6 has yet been
elucidated.
Morikawa et al. (
10) demonstrated that treatment with FOF
in vitro resulted in the suppression of TNF and IL-1 release from
LPS-stimulated human monocytes but an increase in IL-6 release.
Furthermore, Matsumoto et al. (
9) reported that
administration
of FOF to LPS-stimulated mice resulted in reductions in
TNF-
and IL-1
concentrations in serum and slight increases in
IL-6
and IFN-
levels in serum. These results with FOF resembled our
experimental results following the administration of
CLI.
Our present study in the endotoxic shock mouse model suggested that CLI
suppressed the release of inflammatory cytokines,
resulting in a
dose-dependent reduction in mortality. The dose
of CLI used in the
present study was set at a high level, approximately
10 to 20 times the
dose suitable for clinical administration to
humans. Hence, although
immediate clinical application is not
feasible, our results suggest
that CLI may be a useful antibacterial
agent in prevention of septic
shock induced by gram-negative bacilli.
Further studies are necessary
to clarify the mechanism by which
CLI exerts its
effects.
| |
ACKNOWLEDGMENTS |
We thank F. G. Issa (Word-Medex, Sydney, Australia) for the
careful reading and editing of the manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Second
Department of Internal Medicine, Oita Medical University, Hasama, Oita
879-5593, Japan. Phone: 81-97-586-5804. Fax: 81-97-549-4245. E-mail:
mnasu{at}oita-med.ac.jp.
| |
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Antimicrobial Agents and Chemotherapy, September 2001, p. 2638-2642, Vol. 45, No. 9
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.9.2638-2642.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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