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Antimicrobial Agents and Chemotherapy, March 1999, p. 616-622, Vol. 43, No. 3
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Clindamycin Suppresses Endotoxin Released by Ceftazidime-Treated
Escherichia coli O55:B5 and Subsequent Production of
Tumor Necrosis Factor Alpha and Interleukin-1
Kenji
Kishi,
Kazuhiro
Hirai,
Kazufumi
Hiramatsu,
Tohru
Yamasaki, and
Masaru
Nasu*
The Second Department of Internal Medicine,
Oita Medical University, Hasama, Oita, 879-5593, Japan
Received 7 July 1998/Returned for modification 9 November
1998/Accepted 29 December 1998
 |
ABSTRACT |
Treatment of septicemia caused by Escherichia coli with
ceftazidime (CAZ) may be associated with the development of
septic shock due to the release of bacterial lipopolysaccharide. We
examined the suppressive effect of clindamycin (CLDM) on CAZ-induced
release of endotoxin by cultured E. coli and the
subsequent production of inflammatory cytokines (tumor necrosis factor
alpha [TNF-
] and interleukin-1
[IL-1]). E. coli ATCC 12014 was incubated in inactivated horse serum with or
without CLDM for 1, 4, or 18 h, followed by the addition of CAZ
and collection of the culture supernatant at 0, 1, and 2 h. The
concentration of endotoxin in each sample was measured by a chromogenic
Limulus test. Another portion of the culture
supernatant was added to THP-1 cell culture and incubated
for 4 h, and the concentrations of TNF- and IL-1 in the
supernatant were measured by an enzyme-linked immunosorbent assay. In
the control group (no CLDM), CAZ administration resulted in significant
increases in endotoxin, TNF-, and IL-1 concentrations. Pretreatment of E. coli with CLDM for 4 or 18 h
before the addition of CAZ significantly suppressed the concentrations
of endotoxin, TNF-, and IL-1 in a time-dependent manner. In
addition, CAZ treatment transformed E. coli from
rod-shaped bacteria to filament-like structures, as determined by
electron microscopy, while pretreatment with CLDM prevented these
morphological changes. Our in vitro studies showed that CAZ-induced
release of large quantities of endotoxin by E. coli
could be suppressed by prior administration of CLDM.
| |
INTRODUCTION |
Septicemia and septic shock are
life-threatening conditions characterized by fever (sometimes,
hypothermia), organ failure, and low blood pressure. Despite recent
advances in antimicrobial therapy, the mortality rate due to sepsis
caused by gram-negative bacteria remains high at present. For example,
about 300,000 people suffer from septicemia every year in the United
States and about 20 to 30% of these patients die of septic shock
caused by gram-negative bacterial infections (2, 10, 16). In
a survey conducted between January 1993 and April 1994, about 2 of
every 100 hospitalized patients had symptoms related to sepsis, about
40% of these patients had gram-negative bacterial infections, and 25%
of them eventually developed septic shock. In addition, the mortality
rates were 38 and 45% in those patients who were hospitalized for 28 days and 5 months, respectively (23).
An important determinant of mortality in septic shock is the
release of endotoxin. Endotoxin is a structural component
(lipopolysaccharide [LPS]) of the cell wall of gram-negative
bacteria (13, 31). When LPS is released from bacteria,
its active-site lipid A is exposed, leading to the activation of
complement and blood coagulation systems (5, 6, 9, 11, 18, 20,
28). At the same time, macrophages and monocytes are activated
and large quantities of inflammatory cytokines (e.g., tumor
necrosis factor alpha [TNF-] or interleukin-1 [IL-1]) are
released into the circulatory system (17). These changes
trigger a variety of systemic inflammatory reactions mediated by a
complicated cytokine network. Furthermore, in severe septicemia,
several systemic changes lead to circulatory disorders and multiorgan
failure (32).
Although antibiotics are indispensable for the treatment of
septicemia, they may indirectly induce septic shock due to
the rapid lysis of gram-negative bacteria, which enhances the
release of large quantities of endotoxin into the circulatory system
(22). For example, -lactams with a high affinity to
penicillin binding protein 3 (PBP-3) are known to change
the morphology of bacterial cells to a filament form and result
in the release of large quantities of endotoxin (3, 4, 8,
25).
Clindamycin (CLDM) is effective against gram-positive and anaerobic
bacteria, but it is not effective against Escherichia coli.
However, it has recently been reported that subminimal inhibitory concentrations of CLDM may influence bacterial viability, toxin production, and host response. For example, CLDM promotes
phagocytosis of bacteria by neutrophils (29, 30), suppresses
the production of hemolysin by E. coli
(1), inhibits the proinflammatory interactions of
Pseudomonas aeruginosa-derived pigments by neutrophils
(21), and reduces the amount of exotoxin released by
Clostridium perfringens (26). On the other hand,
ceftazidime (CAZ) is an effective antibiotic against E. coli, but when E. coli is killed by CAZ, large
quantities of endotoxin are released (3, 4, 10, 25).
Using cultures of E. coli, we examined the suppressive
effect of CLDM on CAZ-induced release of endotoxin by E. coli and the subsequent production of inflammatory cytokines
(TNF- and IL-1) from THP-1 cells.
| |
MATERIALS AND METHODS |
Organism and preparation of bacterial suspension.
A standard
strain of E. coli ATCC 12014 (E. coli
O55:B5) was used in the present experiments. The organism was stored in
10% skim milk at 
70°C. One day before the experiment, the organism was incubated overnight on Mueller-Hinton II agar (MHA; Becton Dickinson Microbiology Systems, Cockeysville, Md.) at 37°C. One of
the resultant colonies was inoculated in Mueller-Hinton broth (Becton
Dickinson Microbiology Systems) and incubated at 37°C until the
logarithmic growth phase of bacteria was reached. The inoculation dose
of E. coli O55:B5 was adjusted so that the
microorganism was in the logarithmic growth phase (about
107 CFU/ml) following preincubation in the absence of any
antibiotics. Thus, 107, 5 × 105, and
104 CFU/ml were used in 1-, 4-, and 18-h preincubation
experiments, respectively.
Antibiotics.
CAZ (Tanabe Seiyaku, Osaka, Japan) and CLDM
(Pharmacia & Upjohn, Tokyo, Japan) were used in this study. The MICs of
CAZ and CLDM were 0.39 and 200 µg/ml, respectively, as determined by
the standard microdilution procedure recommended by the National
Committee for Clinical Laboratory Standards (19).
Pretreatment with CLDM.
Horse serum (Life Technologies
Oriental, Tokyo, Japan) was inactivated at 58°C for 15 min and used
as the culture medium. CLDM was added to the medium at a concentration
of 25 µg/ml. This concentration is equivalent to that in the
pulmonary tissue of adult patients following a bolus intravenous dose
of 600 mg of CLDM (7). Five milliliters of E. coli O55:B5 inoculum was added to 45 ml of a medium containing
CLDM, and the culture was incubated at 37°C for 1, 4, or 18 h.
Control experiments were also performed by incubation of a similar
volume of E. coli O55:B5 inoculum in inactivated horse
serum without CLDM.
Determination of viable cell counts and endotoxin release.
In the next step, 38 ml of each culture (with or without CLDM) was
divided into two portions and each was placed in a sterile test tube.
In one tube, 1 ml of horse serum containing CAZ (final concentration,
10 µg/ml) was added, while 1 ml of horse serum alone was added in the
other tube. Thus, four experimental groups were prepared: (i) control
group containing E. coli in which no antibiotics were
added, (ii) CAZ group in which E. coli was treated with
only CAZ and not preincubated with CLDM, (iii) CLDM group in which
E. coli was preincubated with CLDM but not treated with CAZ, and (iv) CLDM-CAZ group in which E. coli was
preincubated with CLDM and subsequently treated with CAZ.
Viable cell counts were determined in control, CAZ, CLDM, and CLDM-CAZ
groups at 0, 1, 2, and 4 h after the addition of CAZ by
quantitative cultivation on MHA plates. To determine the concentration of endotoxin, control and test culture samples (5 ml) were collected at
0, 1, or 2 h after the addition of CAZ. Each sample was
centrifuged at 1,000 × g for 15 min at 4°C and
gently subjected to mechanical sterilization with a 0.2-µm-pore-size
filter (EB-DISK 25; Kanto Chemical, Tokyo, Japan). Samples were stored
at 20°C until measurement of endotoxin concentration.
Measurement of endotoxin concentrations.
The concentration
of endotoxin in each sample was measured by a chromogenic
endotoxin-specific assay with Limulus amoebocyte lysate
(LAL) coagulation enzyme. Briefly, 50 µl of each sample was warmed
with 100 µl of perchloric acid solution (Toxicolor system PCA-1 set;
Seikagaku Corp., Tokyo, Japan) for 20 min at 37°C to remove any serum
inhibitory activity against coagulation reaction of LAL. After
centrifugation, the supernatant was neutralized with NaOH solution and
diluted appropriately with endotoxin-free distilled water. Ten
microliters of each diluted solution was mixed with 100 µl of the
main reagent (ES-200 set; Seikagaku Corp.) in each well of a
endotoxin-free 96-well plate (Toxipet Plate 96F; Seikagaku Corp.).
After incubation for 30 min at 37°C, 50 µl each of sodium nitrite
solution, ammonium sulfamate solution, and N-(1-naphthyl)
ethylenediamine dihydrochloride solution were added to complete the
diazo coupling reaction (Toxicolor system DIA-MP set, Seikagaku Corp.),
and A545 of each well was measured with a
spectrophotometer (Multiscan Multisoft; Labsystems Japan K.K., Tokyo,
Japan). Endotoxin levels were calculated relative to the level of the
reference endotoxin, E. coli O111:B4 LPS (Toxicolor system Et-2 set; Seikagaku Corp.).
Production of TNF- and IL-1 by endotoxin-stimulated THP-1
cells.
A human acute monocytic leukemia cell line (THP-1) was used
in the present experiments. THP-1 cells were obtained from a child with
acute monocytic leukemia. This cell line is known to produce TNF-
and IL-1 in response to purified endotoxin (14, 27). The
cells were maintained in RPMI 1640 medium containing 10% inactivated fetal calf serum, 5 × 10
6 M 2-mercaptoethanol, and
0.5% L-glutamine. To induce cytokine production, 2 ml of
THP-1 cells (2 × 106 cells) was seeded into a
pyrogen-free 24-well plate (Nuncron; Nunc, Roskilde, Denmark), and 100 µl of each bacterial filtrate sample was added. The plate was
incubated at 37°C under 5% CO2 for 4 h. The
resultant culture was centrifuged at 250 × g for 2 min
at room temperature, and the supernatant was stored at 20°C until
measurement of TNF- and IL-1.
Direct effect of CLDM on THP-1 cells.
To investigate the
direct effect of CLDM on TNF- production, CLDM was added into the
medium of THP-1 cells (2 × 106 cells) at various
concentrations (3.13, 6.25, 12.5, 25, and 50 µg/ml) or not added
(control). After the cultures were incubated for 1, 4, or 18 h at
37°C under 5% CO2, purified E. coli
O55:B5 LPS (Sigma-Aldrich Corp., Tokyo, Japan) at a final concentration of 1 µg/ml was added to each well and the cells were incubated for
another 4 h. The resultant culture was centrifuged at
250 × g for 2 min at room temperature, and the
supernatant was stored at 20°C until measurement of TNF-.
Measurement of TNF- and IL-1.
The concentrations of
TNF- and IL-1 in the supernatant of THP-1 cell culture were
determined by using an enzyme-linked immunosorbent assay commercial kit
(Cytoscreen; BioSource International, Camarillo, Calif.) according to
the protocols of the manufacturer.
Morphological examination.
Bacterial cultures (100 µl)
were collected from each group before and after 18 h of incubation
with CLDM, as well as 1 h after the addition of CAZ. The sample
was placed on a 0.4-µm-pore-size filter (Isopore Track-Etched
Membrane Filter; Millipore Co., Bedford, Mass.) and fixed first in 2%
glutaraldehyde solution for 1 h and then in 1% osmium tetroxide
for 1 h. The samples were then dehydrated with serial
concentrations of ethyl alcohol (50, 70, 80, 90, 95, and 100%) and
t-butyl alcohol and subjected to critical point drying. The
filter was fixed on a sample table, and after gold palladium coating,
the morphological features of E. coli were examined at ×5,000 magnification with a scanning electron microscope (Hitachi S-800: Hitachi Co., Tokyo, Japan).
Statistical analysis.
For each experiment, the
concentrations of endotoxin, TNF-, and IL-1 were measured in two
wells. All experiments were repeated more than twice on different days.
Differences between values for the groups were tested for statistical
significance by the Mann-Whitney test. A P value of <0.05
denoted the presence of a significant statistical difference.
| |
RESULTS |
Antibacterial activity of CAZ against E. coli.
In a
series of preliminary experiments, we first determined the
antibacterial effects of CAZ against E. coli O55:B5.
For this purpose, about 107 CFU of E. coli
per ml was treated with 2.5, 5, 10, or 20 µg of CAZ per ml (Table
1). Viable cell count was reduced in a
concentration-dependent manner after the addition of CAZ for 4 h
(up to 10 µg/ml).
Effects of CLDM and CAZ on viable cell counts of E. coli.
After preincubation, the viable cell count of
E. coli with CLDM did not significantly decrease
from that of the control, irrespective of the duration of the
preincubation period (Fig. 1). In
addition, further incubation of the control and CLDM groups for 1 to
4 h in the absence of CAZ increased the number of viable bacteria to more than 108 (Fig. 1). In contrast, CAZ resulted in a
rapid decrease in viable cell counts, amounting to 50- to 100-fold in
4 h, in control and CLDM-pretreated cultures (CAZ and CLDM-CAZ
groups [Fig. 1]).
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FIG. 1.
Effects of CLDM and CAZ on viable cell counts of
E. coli O55:B5. E. coli O55:B5 was
pretreated with or without CLDM (25 µg/ml) for 1 (A), 4 (B), or 18 (C) h and then incubated for another 4 h with or without CAZ (10 µg/ml). Symbols: closed circles, control group (no antibiotics used);
closed squares, CAZ group (CAZ treatment only); open circles, CLDM
group (pretreatment with CLDM only); open squares, CLDM-CAZ group
(pretreatment with CLDM followed by CAZ). Data are the means ± standard deviations for four experiments.
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|
Effect of CLDM on CAZ-induced release of endotoxin.
The
concentration of endotoxin released by E. coli after
2 h of incubation was <50 ng/ml in the control and CLDM groups
(Fig. 2). The addition of CAZ to the
culture medium increased the concentration of endotoxin to >1,000
ng/ml at 2 h (Fig. 2). However, pretreatment with CLDM before the
addition of CAZ resulted in suppression of the effect of CAZ (Fig. 2).
This effect was dependent on the duration of preincubation with CLDM.
There was no significant difference in the amount of endotoxin released
by the addition of CAZ following 1 h of pretreatment with CLDM
(1,352 ± 414 versus 1,800 ± 898 ng/ml [Fig. 2A]) compared
to those following CLDM-free pretreatment. However, the amount of
endotoxin released by the addition of CAZ was reduced following 4 h (517 ± 97 versus 1,784 ± 833 ng/ml [P < 0.05] [Fig. 2B]) and 18 h (144 ± 160 versus
1,196 ± 290 ng/ml [P < 0.001] [Fig. 2C]) of
pretreatment with CLDM.
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FIG. 2.
Kinetics of endotoxin released by E. coli O55:B5 after treatment with CAZ. After pretreatment with CLDM
for 1 (A), 4 (B), or 18 (C) h, samples were collected from the control
group (closed circles), CAZ group (closed squares), CLDM group (open
circles), and CLDM-CAZ group (open squares) at 0, 1, and 2 h after
the addition of CAZ and then mechanically sterilized. Data are the
means ± standard deviations for four experiments. Statistical
significance by the Mann-Whitney test is indicated as follows: NS, not
significant (P > 0.05); *, P < 0.05; ***,
P < 0.001.
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|
TNF- and IL-1 concentrations released by THP-1 cells.
In
the control group, the concentrations of TNF- and IL-1 in the
culture supernatant were <100 pg/ml. Incubation with CLDM alone
resulted in small but insignificant changes in the concentrations of
TNF- and IL-1 at 1 and 2 h (Fig.
3 and 4).
In contrast, addition of CAZ significantly increased the concentrations
of TNF- and IL-1 at 2 h (Fig. 3 and 4). However,
pretreatment with CLDM before the addition of CAZ resulted in
suppression of the effects of CAZ (Fig. 3 and 4). This effect was
dependent on the duration of preincubation with CLDM. The
concentrations of TNF- following the addition of CAZ were not
significantly different after 1 and 4 h of pretreatment with CLDM
(957 ± 190 versus 1,171 ± 229 pg/ml and 822 ± 509 versus 1,326 ± 109 pg/ml [Fig. 3A and B, respectively]) compared to those following CLDM-free pretreatment. However, the concentrations of TNF- following the addition of CAZ were reduced after 18 h (266 ± 264 versus 854 ± 88 pg/ml
[P < 0.01] [Fig. 3C]) of pretreatment with CLDM.
In the same manner, the concentrations of IL-1 following the
addition of CAZ were not significantly different after 1 h of
pretreatment with CLDM (494 ± 214 versus 677 ± 192 pg/ml
[Fig. 4A]) compared to those following CLDM-free pretreatment, but
they were reduced after 4 h (367 ± 86 versus 603 ± 15 pg/ml [P < 0.05] [Fig. 4B]) and 18 h
(216 ± 204 versus 537 ± 135 pg/ml [P < 0.05] [Fig. 4C]) of pretreatment with CLDM.
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FIG. 3.
Kinetics of TNF- released by stimulated THP-1 cells.
After pretreatment with CLDM for 1 (A), 4 (B), or 18 (C) h, samples
were collected from the control group (closed circles), CAZ group
(closed squares), CLDM group (open circles), and CLDM-CAZ group (open
squares) at 0, 1, and 2 h after the addition of CAZ and then
mechanically sterilized. THP-1 cells were stimulated by these samples
for 4 h, and the concentration of TNF- in THP-1 cell cultures
was determined. Data are the means ± standard deviations for
four experiments. Statistical significance by the Mann-Whitney test is
indicated as follows: NS, not significant (P > 0.05);
**, P < 0.01.
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FIG. 4.
Kinetics of IL-1 released by stimulated THP-1 cells.
After pretreatment with CLDM for 1 (A), 4 (B), or 18 (C) h, samples
were collected from the control group (closed circles), CAZ group
(closed squares), CLDM group (open circles), and CLDM-CAZ group (open
squares) at 0, 1, and 2 h after the addition of CAZ and then
mechanically sterilized. THP-1 cells were stimulated by these samples
for 4 h, and the concentration of IL-1 in THP-1 cell cultures
was determined. Data are the means ± standard deviations for
four experiments. Statistical significance by the Mann-Whitney test is
indicated as follows: NS, not significant (P > 0.05);
*, P < 0.05.
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On the other hand, 3.13 and 6.25 µg of CLDM per ml did not
significantly reduce the concentration of TNF-
in the medium of
THP-1 cells stimulated with purified
E. coli O55:B5 LPS
regardless
of pretreatment time (Table
2). However, 25 and 50 µg/ml of CLDM
significantly reduced TNF-
concentrations after pretreatment
for 4 or 18 h (Table
2).
Morphological examination.
Morphological examination
showed rod-shaped E. coli cells when not treated
(control) (Fig.
5a). The morphological
features of E. coli were similar to those of the
control after 18 h of preincubation with CLDM (Fig. 5b and c). In
contrast, the addition of CAZ resulted in elongation of the cells
within the first hour (Fig. 5d). However, elongation of the cells was
clearly suppressed when the cells were pretreated with CLDM followed by
the addition of CAZ (CLDM-CAZ group) (Fig. 5e).
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FIG. 5.
Photomicrographs of E. coli O55:B5
examined by electron microscopy. (a) Fresh sample of E. coli before incubation (control), (b) E. coli
after 18 h of preincubation in CLDM-free medium, (c) E. coli after 18 h of preincubation with 25 µg of CLDM per ml,
(d) E. coli preincubated in CLDM-free medium shown
1 h after the addition of 10 µg of CAZ per ml, and (e)
E. coli preincubated with 25 µg of CLDM per ml shown
1 h after the addition of 10 µg/ml of CAZ. Note transformation
of E. coli to filament-like structures in panel d.
Bars, 3 µm.
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|
| |
DISCUSSION |
The major finding of this study was that pretreatment of cultured
E. coli with CLDM for 4 to 18 h reduced the amount
of CAZ-induced increase in bacterial endotoxin levels and prevented the
associated rise in TNF- and IL-1 production. We also showed that
these changes were associated with elongation of the bacteria. The
mechanisms of CLDM-induced suppression of CAZ-enhanced
endotoxin release are not clear at present but may be due, at least in
part, to the morphological changes described above. It is possible that the suppression of filament formation reduces the surface area of lysed
bacteria, which in turn decreases the amount of endotoxin released by
E. coli. Several studies have reported that -lactam antibiotics with a high affinity to PBP-3 (e.g., CAZ) enhance elongation of gram-negative bacteria and that due to subsequent bacteriolysis, large quantities of endotoxin are released in the supernatant during the early phase of incubation (3, 4, 25).
Jackson and Kropp (8) compared the effects of two -lactam antibiotics (imipenem [IPM] and CAZ that show a strong affinity toward PBP-2 and PBP-3, respectively) on P. aeruginosa and
reported that CAZ released 10 to 40 times more endotoxin than IPM. In
addition, Dofferhoff et al. (3, 4) examined the effects of
various antibiotics on E. coli cultured in a solution
containing human monocyte cells by measuring the concentrations of
endotoxin and TNF-. They reported higher concentrations of endotoxin
and TNF- in cultures treated with low concentrations of
CAZ, aztreonam, and cefuroxime than in cultures treated with IPM. Simon
et al. (25) also reported results similar to those described
above with THP-1 cells, which we used in this study. They reported that incubating these cells with supernatant of E. coli
treated with CAZ, cefotaxime, or aztreonam was associated with higher
concentrations of TNF- than in THP-1 cells treated with a
supernatant of E. coli treated with IPM or no antibiotics.
Our results also showed a fast bactericidal effect for CAZ against
E. coli. CAZ rapidly killed E. coli
O55:B5, and more than 1,000 ng/ml of endotoxin was released after
2 h of incubation. In addition, there was no significant
difference in viable cell count between CAZ and CLDM-CAZ groups, i.e.,
25 µg of CLDM per ml did not affect the growth of E. coli or the bactericidal activity of CAZ. Furthermore, 18 h
of preincubation with CLDM did not change the amount of endotoxin
released by E. coli. In contrast, the addition of CAZ
enhanced the production of endotoxin to more than 1,000 ng/ml. However,
pretreatment of such cultures with CLDM for 4 and 18 h
significantly reduced the amount of endotoxin to 517 and
144 ng/ml, respectively. Similar changes were also observed for TNF- and IL-1 released into the supernatant; the
concentrations of both cytokines were higher following CLDM-free
pretreatment than following pretreatment with CLDM for 4 and 18 h.
Previous studies have shown that CLDM suppresses the production of
-toxin by C. perfringens (26) and hemolysin by
E. coli (1). These toxins are proteins, but
endotoxin itself is a LPS. Nonetheless, due to the suppression of
enzymes that inhibit the synthesis of cell walls, the amount of
endotoxin may be secondarily decreased.
CLDM is also known to enhance the cytoplasmic function of
polymorphonuclear leukocytes (11), accelerate phagocytosis
(24), promote antibody production by lymphocytes
(15), and suppress the production of superoxide and
myeloperoxidase in human neutrophils when stimulated by pyocyanin or
1-hydroxyphenazine, both of which are produced by P. aeruginosa (21). These findings suggest that CLDM is an
effective immunomodulator. In fact, Stevens et al. (26)
reported that CLDM suppressed the production of TNF- by LPS-stimulated human monocytes. In the present study, 3.13 µg of CLDM
per ml failed to suppress the production of TNF- when THP-1 cells
were stimulated with purified LPS. This level of CLDM was larger than
the level in the bacterial filtrate sample added into the THP-1 cell
culture in this study. Nevertheless, 25- and 50-µg/ml
concentrations of CLDM, which are attainable in pulmonary tissue
clinically (7), significantly reduced TNF- concentrations after pretreatment for 4 or 18 h. Therefore, excess production of
inflammatory cytokines such as TNF- induced by release of LPS
endotoxin may be suppressed.
In conclusion, we have shown that pretreatment with CLDM
suppresses CAZ-enhanced release by E. coli of
endotoxin, TNF-, and IL-1. From these findings, we intend to
study further the suppressive effect of CLDM against endotoxin
release by gram-negative bacteria in in vitro and in vivo models.
| |
ACKNOWLEDGMENTS |
We thank F. G. Issa from the Department of Medicine,
University of Sydney, Sydney, Australia, for the careful reading and editing of the manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: The 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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Abstract
Introduction
