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Antimicrobial Agents and Chemotherapy, November 2000, p. 3169-3173, Vol. 44, No. 11
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Protection against Lipopolysaccharide-Induced Death
by Fluoroquinolones
Anis A.
Khan,1,2
Teri R.
Slifer,1
Fausto G.
Araujo,1
Yasuhiro
Suzuki,1,2 and
Jack S.
Remington1,2,*
Department of Immunology and Infectious
Diseases, Research Institute, Palo Alto Medical Foundation, Palo Alto,
California 94301,1 and Division of
Infectious Diseases and Geographic Medicine, Department of Medicine,
Stanford University School of Medicine, Stanford, California
943052
Received 1 February 2000/Returned for modification 15 May
2000/Accepted 5 August 2000
 |
ABSTRACT |
Because fluoroquinolones have an immunomodulatory effect on
cytokine production by lipopolysaccharide (LPS)-treated human monocytes, we examined the effect of fluoroquinolones on the survival of mice injected with a lethal dose of LPS. Trovafloxacin (100 mg/kg),
ciprofloxacin (250 mg/kg), and tosufloxacin (100 mg/kg) protected 75%
(P = 0.0001), 25% (P = 0.002), and
50% (P = 0.002), respectively, of mice against death.
The fluoroquinolones significantly reduced serum levels of
interleukin-6 and tumor necrosis factor alpha in LPS-treated mice. The
protective effects of fluoroquinolones in LPS-induced shock in mice may
also occur in humans.
| |
TEXT |
Septic shock that results from
gram-negative bacterial infections is a major cause of death among
patients in intensive care units (17). The presence of
bacterial lipopolysaccharide (LPS) in the bloodstream causes fever,
hypotension, multiple organ failure and, in severe cases, septic shock
and death (16). Tumor necrosis factor (TNF) and
interleukin-1 (IL-1) have been shown to be important mediators of
shock; both induce changes that are similar to those induced by
endotoxin or live gram-negative bacteria (7, 18). Although
concentrations of these cytokines fluctuate during sepsis (21), TNF is elevated in the sera of patients in septic
shock, and most studies have shown an association between TNF levels, severity of shock, and fatality. The serum levels of IL-1, IL-6, and
IL-10 are also frequently elevated in these patients. High peak levels
or persisting elevation of IL-6 levels has been stated to be a
predictor of adverse outcome (21).
TNF, IL-1, and platelet-activating factor have been identified as
important mediators in development of inflammation induced by sepsis
(2, 4, 11). However, clinical trials in humans with sepsis
or septic shock with TNF-
blocking antibodies (1, 8),
IL-1 receptor antagonists (12), and platelet-activating factor receptor antagonists (10) have been unsuccessful.
Also, attempts to downregulate the inflammatory process by using
methylprednisolone (6) or to prevent the initiation of
sepsis by using monoclonal antibodies directed against endotoxin
(5, 14, 22) have failed to show benefit of these treatment modalities.
We have previously shown that trovafloxacin reduces the in vitro
production of TNF-, IL-1
, and IL-6 by LPS-stimulated human monocytes (15). In the present study, we considered it of
interest to determine whether three structurally related
fluoroquinolones
trovafloxacin, ciprofloxacin, and
tosufloxacinaffect serum concentrations of cytokines and prevent or
reduce mortality in mice injected with a lethal dose of LPS.
Antibiotics.
Trovafloxacin (Pfizer, Inc., Groton, Conn.),
ciprofloxacin (Bayer Corp., West Haven, Conn.), and tosufloxacin
(Abbott Laboratories, Chicago, Ill.) were dissolved as directed by the
supplier and administered orally by gavage.
Mice.
Outbred Swiss Webster (SW) and inbred BALB/C female mice
weighing approximately 20 g at the beginning of each experiment
were purchased from Taconic Farms, Inc. (Germantown, N.Y.) and given water and food ad libitum.
Effect of antibiotics on mortality induced by LPS.
In
preliminary experiments, doses of 10 or 100 µg of LPS
(Escherichia coli O111:B4; Sigma Chemical Co., St. Louis,
Mo.) administered intravenously (i.v.) resulted in 100% survival.
Mortality following a dose of 500 µg varied from 50 to 100%; a
1,000-µg dose always resulted in 100% mortality. The effect of
pretreatment with antibiotics on mortality was investigated by treating
SW mice orally with doses of 100 or 200 mg of trovafloxacin, 100 or 250 mg of ciprofloxacin, or 100 or 200 mg of tosufloxacin per kg at 47, 17, and 1 h prior to the injection of 1,000 µg of LPS. Control mice
received antibiotics alone or LPS alone. Mice were observed daily at
8 a.m. and 5 p.m., and mortality was recorded for 1 week
after the injection of LPS. The Kaplan-Meier product limited-survival
analysis was used to analyze the survival data (StatView, version 4.02;
Abacus Concepts, Berkeley, Calif.). A P value of 
0.05 was
considered significant. A series of 11 experiments with 6 to 10 SW mice
in each treatment group revealed comparable results. The results of
representative experiments are shown in Fig.
1. Treatment with 100 or 200 mg of
trovafloxacin per kg resulted in survival of 75% (Fig. 1A) and 67% of
the mice, respectively (P = 0.001), whereas treatment with 250 mg of ciprofloxacin per kg resulted in survival of 25% of the
mice (P = 0.008) (Fig. 1B). Treatment with a 100-mg/kg dose did not afford significant protection against death. An experiment of the same design in BALB/c mice (10 mice in each group) using 250 mg
of ciprofloxacin per kg resulted in 30% survival (P = 0.001). Survival rates following treatment of SW mice with 100 or
200 mg of tosufloxacin per kg were 50% (P = 0.003) and
30% (P = 0.008), respectively (six mice in each
group).
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FIG. 1.
Survival in SW mice treated orally with 100 mg of
trovafloxacin per kg and injected i.v. with 1,000 µg of LPS 1 h
later (A) or treated orally with 250 mg of ciprofloxacin per kg and
injected with LPS 1 h later (B). The P values, as
determined by Kaplan-Meier product limited-survival analysis, were
0.001 for trovafloxacin and 0.008 for ciprofloxacin.
|
|
In separate experiments to determine whether a single dose of
antibiotics administered 1 h before 1,000 µg of LPS would also
be protective, mice were treated with 100 or 200 mg of trovafloxacin
per kg 1 h before injection of LPS. The results of a
representative
experiment of the three performed revealed that 17%
(
P = 0.010)
of the mice treated at 100 mg/kg and 33%
(
P = 0.0006) of the mice
treated at 200 mg/kg survived
longer than the controls injected
with LPS alone (10 mice in each
group).
In five experiments performed to determine whether administration of
the antibiotics after the injection of LPS also would
be protective,
groups of 5 to 10 SW mice each were injected i.v.
or intraperitoneally
with 500 or 1,000 µg of LPS and, 1 h or 10
min later, were
treated orally with 100 mg of trovafloxacin or
250 mg of ciprofloxacin
per kg. The results varied from an earlier
death in the LPS-antibiotic
treated mice than in controls that
received only LPS to 25 and 30%
survival rates in mice treated
with trovafloxacin or ciprofloxacin,
respectively. The reasons
for this variation are unclear. Mice appeared
ill 1 h following
LPS injection, and by 3 h they were huddled
together and lethargic.
Attempts to administer a second dose of the
antibiotics at 6 h
after the first dose were not
successful.
Effect of antibiotics on serum levels of cytokines.
Determination of serum levels of cytokines was conducted using
commercially available enzyme-linked immunosorbent assay reagents (PharMingen, San Diego, Calif., and Endogen, Woburn, Mass.).
Quantification was based on a standard curve derived by linear dilution
of the cytokine standards included in the respective kits. The
detection limits were as follows: 4 pg/ml for IL-2; 8 pg/ml for
IL-1, IL-4, IL-6, IL-10, and TNF-; and 20 pg/ml for IL-12 (p40)
and gamma interferon (IFN-
). Each assay was performed in duplicate
wells. Statistical analysis of the differences in cytokine levels was determined using Welch's modified t test. A P value of
0.05 was considered significant.
Initial experiments were performed to determine the effects of 100 mg
of either trovafloxacin alone, ciprofloxacin alone,
or LPS alone per
kg. Controls were as described in Table
1. Mice
were bled by cardiac puncture
under anesthesia at 1 and 3 h after
treatment. The serum levels of
each of the cytokines were significantly
elevated at 1 and 3 h
following injection of LPS compared with
sham-treated controls. The
combined results of two separate experiments,
with 10 mice in each
group, are shown in Table
1. Treatment with
trovafloxacin resulted in
significant increases in the levels
of IL-2, IL-4, IL-10, and TNF-
at 1 and 3 h, of IL-6 and IL-12
at 1 h, and of TNF-
at
3 h (Table
1). Treatment with ciprofloxacin
resulted in
significant increase in levels of IL-2, IL-4, and
TNF-
at 1 and
3 h, of IL-12 at 1 h, and of IL-10 and TNF-
at
3 h.
Although the increases in levels of cytokines were significantly
higher
in mice treated with antibiotics than in sham-treated controls
(injected with diluent), they were not significantly higher than
those
noted in mice injected with LPS alone (Table
1).
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TABLE 1.
Cytokine levels in blood of mice at 1 and 3 h after
administration of LPS, ciprofloxacin, or trovafloxacin
|
|
The effect of prior treatment with trovafloxacin, ciprofloxacin, or
tosufloxacin on serum cytokine levels was examined 1 and
4 h after
injection of LPS. Treatment with trovafloxacin resulted
in a
significant reduction in the levels of IL-6 and TNF-
(
P = 0.009 and
P = 0.01, respectively) at 1 h
after LPS injection
(Fig.
2A) and of IL-1
and TNF-
at 4 h (
P = 0.04 and
P = 0.003,
respectively) (Fig.
2B). Treatment with ciprofloxacin
resulted
in significant reduction in levels of IL-6 and TNF-
(
P = 0.02
and
P = 0.05, respectively)
1 h after LPS injection (Fig.
3A);
only the levels of TNF-
were significantly reduced (
P = 0.05)
at 4 h (Fig.
3B). Results with tosufloxacin were
similar to those
noted with ciprofloxacin (data not shown). The serum
levels of
each of the other cytokines were not significantly different
from
those of the controls.
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FIG. 2.
Levels of serum cytokines in mice treated orally with
100 mg of trovafloxacin per kg and injected i.v. with 1,000 µg of LPS
at 1 h (A) and 4 h (B) after injection. *, P
values of 0.05 compared with LPS alone.
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FIG. 3.
Levels of serum cytokines in mice treated orally with
100 mg of ciprofloxacin per kg and injected i.v. with 1,000 µg of LPS
at 1 h (A) and 4 h (B) after injection. *, P
values of 0.05 compared with LPS alone.
|
|
The in vitro immunomodulatory activity of fluoroquinolones has been
described by a number of investigators using both mouse
and human
immune cells (
3,
15,
19). The results described
above reveal
that oral treatment with trovafloxacin, ciprofloxacin,
or tosufloxacin
in mice prior to the induction of a lethal LPS-induced
septic
shock-like state significantly protects against death.
Significant
protection was conferred even when a single dose of
trovafloxacin was
administered 1 h before
LPS.
The mechanism(s) by which these antibiotics protect LPS-injected mice
against death is not clear. Following treatment with
the
fluoroquinolones alone, each of the cytokines tested except
for IL-1
was upregulated in mice. This, along with the downregulation
of IL-6
and TNF-
observed after administration of LPS to the
antibiotic
treated mice, may, at least partially, have been responsible
for their
protective activity. Although cytokine responses vary
considerably in
patients with sepsis, the proinflammatory cytokine
network and lack of
appropriately modulated anti-inflammatory
mediators are considered
central to the pathophysiology of the
sepsis syndrome (
9,
13,
20).
The doses of trovafloxacin used in the present studies were higher than
and those of ciprofloxacin were comparable to those
used in humans when
corrected for the per-square-meter surface
area of mice and humans.
Blood and tissue levels in mice given
these antibiotics by gavage were
not performed and were beyond
the scope of this study. Our results
reveal that each of the three
fluoroquinolones significantly reduced
LPS-induced mortality in
mice and modulated mediator cytokines in vivo.
The protective
effects of fluoroquinolones in LPS-induced shock in mice
may also
occur in
humans.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Immunology and Infectious Diseases, Research Institute, Palo Alto
Medical Foundation, Ames Bldg., 795 El Camino Real, Palo Alto, CA
94301. Phone: (650) 853-6061. Fax: (650) 329-9853.
| |
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Antimicrobial Agents and Chemotherapy, November 2000, p. 3169-3173, Vol. 44, No. 11
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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