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Lovelace Respiratory Research Institute, Albuquerque, New Mexico 87185
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ABSTRACT |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Interleukin
(IL)-10 inhibits the synthesis of proinflammatory cytokines implicated
in fever, including IL-1
, IL-6, and tumor necrosis factor (TNF)-
.
We hypothesized that IL-10 functions as an antipyretic in the
regulation of fevers to lipopolysaccharide (LPS) and turpentine. Body
temperature was measured by biotelemetry. Swiss Webster (SW) mice
treated with recombinant murine IL-10 were resistant to fever induced
by a low dose of LPS (100 µg/kg ip) and to the hypothermic and
febrile effects of a high (septiclike) dose of LPS (2.5 mg/kg ip).
IL-10 knockout mice developed an exacerbated and prolonged fever in
response to a low dose of LPS (50 µg/kg ip) compared with their
wild-type counterparts. At 4 h after injection of the low dose of LPS,
plasma levels of IL-6, but not TNF-, were significantly elevated in
the IL-10 knockout mice compared with their wild-type controls (ANOVA,
P < 0.05). After injection of the
same high dose of LPS injected into SW mice, wild-type mice developed a
fever at 24 h whereas IL-10 knockout mice immediately developed a
profound hypothermia that lasted through 41 h (ANOVA, P < 0.05). Body weight and food
intake were more significantly depressed in response to the high dose
of LPS in the knockout mice compared with their wild-type controls.
Only 30% of the IL-10 knockout mice survived compared with 100% of
the wild-type mice (Fisher's exact test,
P < 0.05). Fever in response to the
injection of turpentine (100 µl/mouse sc) did not differ between
wild-type and IL-10 knockout mice. These data support the hypotheses
that 1) IL-10 functions as an
endogenous antipyretic following exposure to LPS,
2) a putative mechanism of the early
antipyretic action of IL-10 is through the inhibition of plasma levels
of IL-6, 3) IL-10 has a protective
role in the lethal effects of exposure to high levels of LPS, and
4) endogenous IL-10 does not have a role in fever induced by turpentine.
acute phase response; anorexia; hypothermia; temperature regulation; sepsis; lipopolysaccharide
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INTRODUCTION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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A FEVER IS a regulated rise in body temperature (Tb) resulting from contact with infectious or inflammatory stimuli. Although there are considerable data indicating that moderate fevers are beneficial to the infected host, above a certain temperature (e.g., 41°C) the elevation in Tb, by itself, may be harmful (14). Fortunately fevers seldom reach these dangerous levels, an observation made about 50 years ago by DuBois (5).
Many cytokines are capable of modulating fever and are important for
the orchestration of both systemic and local inflammatory responses.
Interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF)- have
been implicated as key mediators of fever using several animal models.
IL-1, IL-6, IL-8, macrophage inflammatory protein-1, interferon
(IFN)-
, and others have been characterized as endogenous pyrogens,
or fever-inducers (13). Many hormones are also endogenous antipyretics;
that is, they reduce fever. These include -melanocyte-stimulating hormone, arginine vasopressin, and glucocorticoids (12, 26, 32). There
is evidence that TNF- may be an endogenous pyrogen under certain
circumstances and an endogenous antipyretic under other conditions (15,
19, 21).
IL-10 is a protein product of T helper (Th) 2 subset cells that was
originally described as a "cytokine synthesis inhibitory factor."
IL-10 suppresses Th1-dependent IL-2 and IFN- production and, in
turn, proliferation of Th1 cells (7). IL-10 also inhibits the synthesis
of a number of proinflammatory cytokines implicated in the regulation
of fever, including TNF-, IL-1, IL-1, IL-6, and IL-8 following
in vitro stimulation of monocytes by bacterial lipopolysaccharide (LPS;
active component of gram-negative endotoxin; Refs. 7, 11). In addition,
IL-10 increases IL-1 receptor antagonist production from LPS-stimulated
neutrophils (11).
Injection of LPS is a widely used experimental model of systemic
inflammation and is a major causative agent in the pathogenesis of
septic shock. Elevations of plasma IL-10 levels have been detected in
patients with sepsis and after the injection of LPS into experimental animals (6, 24). Suppression of TNF-, IL-8, IL-12, and IFN- following injection of IL-10 during septic shock is thought to be
responsible for the protection afforded by IL-10 in endotoxemic mice
and primates (8, 33, 35). Interestingly, it was recently demonstrated
in rats that the central injection of IL-10 inhibits fever in response
to LPS injection (27).
Turpentine is a widely used model of local inflammation. Using gene
knockout mice, our laboratory has demonstrated a key role for IL-1
and IL-6 in fever induced by the subcutaneous injection of turpentine
(16, 38). On the other hand, TNF- does not appear to mediate the
febrile response to turpentine in mice (21). To our knowledge, the role
of IL-10 in fever induced by turpentine has not been investigated.
The aim of this study was to examine the role of IL-10 in fever and
cytokine release induced by systemic (i.e., LPS) and local (i.e.,
turpentine) inflammatory stimuli. On the basis of the ability of IL-10
to inhibit the production of IL-1, IL-6, and TNF- (7, 11), we
hypothesized that the pharmacological action of IL-10 would be an
attenuation of fever in response to the peripheral injection of LPS. In
addition, we used IL-10 knockout mice to test the hypothesis that IL-10
functions as an endogenous antipyretic. We hypothesized that IL-10
knockout mice would develop exacerbated febrile responses to LPS and
turpentine compared with their wild-type controls. To induce systemic
inflammation, we injected low (50 or 100 µg/kg ip) or high,
septiclike (2.5 mg/kg ip) doses of LPS into Swiss Webster (SW) or IL-10
knockout mice. For the induction of local inflammation, mice were
injected with a single dose of turpentine (100 µl/mouse sc). We
demonstrated that whereas IL-10 functions as an endogenous antipyretic
in the regulation of fevers induced by LPS, it may not have a
physiological role in the regulation of
Tb in response to a local
inflammation induced by turpentine in mice. Furthermore, inhibition of
plasma concentrations of IL-6 in response to a low dose of LPS is a
putative mechanism of the antipyretic action of IL-10 in mice.
Because we have shown previously in our laboratory that sepsis induced by the injection of LPS or cecal ligation and puncture (CLP) induces large reductions in body weight and food intake of mice (21, 22), we also examined changes in these variables in the wild-type and IL-10 knockout mice injected with the high dose of LPS. IL-10 knockout mice developed significantly larger reductions in body weight and food intake in response to the high dose of LPS compared with their wild-type counterparts. These differences were paralleled by significant differences in the lethality of sepsis in the wild-type (100% survival) and IL-10 knockout mice (30% survival).
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MATERIALS AND METHODS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Experimental animals. For recombinant murine IL-10 (rmuIL-10) injection experiments, specific pathogen-free adult male SW mice weighing 30-35 g were purchased from Taconic Laboratories (Germantown, NY). Additional experiments used adult specific pathogen-free male C57BL/10 wild-type and IL-10 knockout mice weighing ~30-35 g purchased from Jackson Laboratories (Bar Harbor, ME). The original generation of the IL-10 knockout mice and the strategy for inactivation of the IL-10 gene by homologous recombination are described in detail elsewhere (18). Although the development of chronic enterocolitis and other abnormalities have been reported in the IL-10 knockout mice, we did not observe any phenotypic, behavioral, or Tb abnormalities in these mice before experimentation.
All mice were housed one per cage in a room maintained at 30°C, with a 12:12-h light-dark cycle (lights on at 0600) and humidity controlled to 30-40%, and provided with ad libitum tap water and laboratory rodent chow (Teklad Rodent Diet W8604). All mice were housed in a facility approved by the American Association for Accreditation of Laboratory Animal Care.
Measurement of Tb. Core Tb (±0.1°C) was monitored using the Datacol 3 biotelemetry system (Mini-Mitter, Sunriver, OR). Animals were anesthetized with halothane for the intraperitoneal surgical implantation of the Mini-Mitter transmitters (coated with dental wax) and were allowed 7 days to recover from this surgery before the onset of experimentation. The transmitters sent signals whose frequencies vary consistently with Tb to receiver boards located under the cages. The receiver boards were connected to an IBM-PC that processed the frequency signals into temperatures using predetermined calibration values. This system allowed the temperature of undisturbed animals to be monitored at 5-min intervals throughout all experiments, beginning at least 24 h before the experimental procedures and continuing until the animals were euthanized. The transmitters were recalibrated after each experiment to ensure that recorded temperatures were accurate. Data from a transmitter that did not recalibrate to within ± 0.1°C of the preimplantation value were excluded from all analysis and presentation.
LPS. Purified lyophylized extract of Escherichia coli endotoxin (0111:B4; Sigma Chemical, St. Louis, MO) was dissolved in 0.9% sodium chloride (saline) to a stock concentration of 2 mg/ml and stored at
20°C. Before injection, the stock solution was warmed to
37°C, diluted in sterile saline to the desired concentration, and
intraperitoneally injected at a low dose of 50 µg/kg (C57BL/10 and
IL-10 knockout mice) or 100 µg/kg (SW mice) or at a high dose of 2.5 mg/kg (all mice). Control mice were intraperitoneally injected with an
equivalent volume of sterile saline. Injection volume did not exceed
0.15 ml/mouse. All injections were performed between 0900 and 0930.
Turpentine. Commercial-grade
steam-distilled turpentine (Sunnyside, Wheeling, IL) was injected
subcutaneously into the left hindlimb at a volume of 100 µl/mouse.
Control mice received 100 µl of sterile saline subcutaneously into
the same injection site. All mice were briefly anesthetized with
halothane to relieve the pain of the injection procedure. All
injections were performed between 0900 and 0930.
rmuIL-10. rmuIL-10
(E. coli) was a generous gift from
Dr. Grace H. W. Wong (Department of Molecular Biology, Genentech, South San Francisco, CA). rmuIL-10 was injected intraperitoneally into SW
mice at a concentration of 1.0 µg/mouse (~30 µg/kg). Control mice
were intraperitoneally injected with an equivalent volume of sterile
saline. Injection volume did not exceed 0.15 ml/mouse. All injections
were performed between 0900 and 0930. rmuIL-10 was injected immediately
before sterile saline or LPS.
Blood collection for cytokine
measurements. Blood for cytokine analyses was collected
from anesthetized C57BL/10 and IL-10 knockout mice by cardiac puncture
at 1, 4, and 24 h after injection of sterile saline (equivalent volume)
or low-dose LPS (50 µg/kg ip). Blood was drawn into heparinized
syringes to determine plasma IL-6- and TNF--like activity. Plasma
was separated by centrifugation of the freshly drawn blood and stored
at 20°C. Because of a limited supply of rmuIL-10, we were
unable to assess plasma cytokine levels in SW mice.
Bioassay for IL-6. The IL-6-dependent
mouse B9 hybridoma cell line was used to determine IL-6 activity in
plasma. The B9 line was kindly provided by Dr. Lucien Aarden,
Amsterdam, The Netherlands (1). Plasma samples to be tested for IL-6
were serially diluted in 10% fetal calf serum, 2 mM
L-glutamine, 100 U/ml
penicillin, and 10 µg/ml streptomycin in Iscove's modified
Dulbecco's medium (Sigma), and 100-µl volumes were added in
triplicate to 96-well tissue culture plates. Cells were washed and
resuspended at 5 × 104
cells/ml in growth medium supplemented with 10 U/ml sterile heparin, and 100 µl of cell suspension were added to each well. The plates were incubated for 64-68 h at 37°C, 5%
CO2, and 98% humidity. Then, 20 µl of 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT; 5 mg/ml, Sigma) were added to each well to determine the amount
of proliferation (MTT is a tetrazolium salt that will form dark
crystals when combined with metabolizing cells). After being allowed to
incubate for an additional 4 h, 150 µl of supernatant were removed
from each well, and 100 µl of 10% SDS (Sigma) in 0.01 N HCl were
added to dissolve the tetrazolium crystals. The plates were protected
from light and left overnight at room temperature before being read at
570 and 630 nm (for reference) using an EL312 Bio-Kinetics reader
(Bio-Tek Instruments, Winooski, VT). Units were then calculated based
on a purified human recombinant (hr) IL-6 standard
[1 IU = 0.01 ng; First International Standard Code 89/548,
Stephen Poole, National Institute for Biological Standards and Control,
UK (NIBSC)] run in the same assay. From this standard curve, a
best-fit line was used to calculate the IL-6 activity in the samples.
The sensitivity of the bioassay was 0.5-1.0 IU/ml. Because we did
not specifically neutralize the activity of the proliferative factor
with IL-6 blocking antibodies, the reported values are considered
"IL-6-like" activity.
Bioassay for TNF-. The TNF-
bioactivity in plasma was determined using lysis of WEHI 164 subclone
13 cell line (gift from Dr. Anders Waage, University of Trondheim,
Norway) as described elsewhere (23). Briefly, cells (50 × 104/ml) were incubated (37°C,
5% CO2, and 98% humidity) in the
presence of 0.5 µg/ml actinomycin D, 10 U/ml sterile heparin, and 100 µl of serial dilutions of the test samples. After 18-20 h, 20 µl MTT (5 mg/ml; Sigma) were added to determine the amount of
remaining WEHI cells and allowed to incubate for an
additional 4 h. Then, 150 µl of supernatant were removed and 100 µl
10% SDS (Sigma) in 0.01 N HCl were added to each well. The plates were
left overnight at room temperature to dissolve the crystals. On the
following day, the plates were read at 570 and 630 nm (for reference)
using an EL312 Bio-Kinetic reader (Bio-Tek Instruments, Winooski, VT), and the units of TNF were calculated based on an hrTNF standard curve
(1 IU = 0.025 ng, First International Standard Code 87/650, NIBSC) run
in the same assay. The best-fit line of the standard curve was used to
calculate the TNF bioactivity in the samples. Sensitivity of the assay
was 0.5-1.0 IU/ml. Because we did not neutralize the measured TNF
activity in the plasma with TNF blocking antibodies, the reported
values are considered "TNF-like" activity.
Statistical analysis. Results are
presented as means ± SE. To test for statistical significance,
Tb, changes in body weight and
food intake, and plasma cytokine values were compared between groups by
one-way ANOVA followed by post hoc Scheffé's test or Fisher's
protected least-significant difference (bioassays only). Survival data
were analyzed by Fisher's exact test. A value of P < 0.05 was considered significant.
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Effect of rmuIL-10 on fever induced by a low dose of LPS in SW mice. Figure 1 depicts 1-h averages of Tb in SW mice injected with rmuIL-10 (1 µg/mouse ip), immediately followed by a low dose of LPS (100 µg/kg ip). Before injection at 0 h, all SW mice had virtually identical Tb. The transient increase in Tb seen in all groups of mice at 0 h represents a stress-induced elevation due to handling during the injection procedure. SW mice injected with rmuIL-10 alone showed the normal circadian rhythm in Tb, with low daytime and high nighttime values. Mice intraperitoneally injected with LPS responded with an ~0.80°C monophasic fever that peaked at ~5 h. Pretreatment of mice with rmuIL-10 resulted in a significant attenuation of LPS-induced fever from 3 to 10 h (P < 0.05). Tb did not differ between groups during the nighttime hours (11-21 h).
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Effect of rmuIL-10 on fever induced by a high dose of
LPS in SW mice. Figure 2
depicts the 1-h averages of Tb of
SW mice injected with rmuIL-10 (1.0 µg/mouse ip) immediately before
the injection of a high dose of LPS (2.5 mg/kg ip). All mice had
virtually identical Tb before
injections at 0 h. As shown previously, the intraperitoneal injection
of rmuIL-10 alone had virtually no effect on the normal circadian
rhythm of Tb of SW mice
(Fig. 2). The transient rises in
Tb at 0 and 24 h are
stress-induced rises due to injection and weighing procedures,
respectively. Injection of the large, septiclike dose of LPS resulted
in a biphasic pattern of Tb that differed from the fever induced by 100 µg/kg (compare Figs. 1 and 2).
Mice injected with this high dose of LPS had an initial drop of
Tb that peaked at ~4 h, lasted
throughout the first day of injection, and has been shown previously to
be associated with the presence of high circulating levels of
biologically active TNF- (4, 15).
Tb increased during the first
night following injection and resulted in an ~0.90°C fever by 24 h postinjection compared with preinjection
Tb
(P < 0.05). Due to the
stress-induced rise in Tb as a
consequence of weighing at 24 h, this peak in fever represented only an
~0.5°C rise above the Tb of
the animals injected with rmuIL-10 alone.
Tb of the group injected with LPS alone was afebrile during the second night after injection. The pretreatment of SW mice with rmuIL-10 significantly attenuated both the
hypothermic (2-9 h; P < 0.05)
and febrile phase (16-33 h; P < 0.05) of the Tb response to this
high dose of LPS.
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Fever in response to a low dose of LPS in IL-10 knockout mice. To examine the effect of the elimination of endogenous IL-10 signaling on the febrile response elicited to a low dose of LPS, we used IL-10 knockout mice of C57BL genetic background (18) and their sex- and age-matched wild-type counterparts. Before injection, all groups of mice had virtually identical Tb (Fig. 3). Wild-type and IL-10 knockout mice injected with sterile saline showed similar biphasic circadian Tb, with low daytime and high nighttime values, indicating that endogenous IL-10 is not a critical regulator of the normal circadian rhythm of Tb. Wild-type mice injected intraperitoneally at 0 h with LPS at a dose of 50 µg/kg developed an ~1.6°C fever that was significantly elevated above their saline-injected controls from 1 to 5 h during the first day after injection (P < 0.05). Starting at 11 h and lasting until the end of the experimental observation period, wild-type mice injected with LPS had virtually identical Tb responses as their saline-injected controls. Thus this low dose of LPS elicited a relatively transient (~5 h) fever in the wild-type mice. IL-10 knockout mice injected with the same dose of LPS showed an exacerbated and prolonged febrile response. During the first day following injection, LPS-injected knockout mice developed a fever that was significantly elevated above their LPS-injected wild-type controls from 4 to 11 h (P < 0.05). IL-10 knockout mice manifested an additional febrile response the second day after injection from 20 to 28 h that was absent in the LPS-injected wild-type mice (P < 0.05). Subsequent to this latter time point, wild-type and IL-10 knockout mice injected with LPS maintained similar Tb profiles.
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Plasma IL-6 and TNF- levels in IL-10 knockout mice
injected with a low dose of LPS. Plasma IL-6- and
TNF--like activity was measured in wild-type and IL-10 knockout mice
at 1, 4, and 24 h after injection of a low dose of LPS (50 µg/kg ip)
or sterile saline (equivalent volume). At all time points tested after
injection of sterile saline, wild-type and IL-10 knockout mice showed
low plasma IL-6-like activity that did not differ between groups (Fig. 4A).
Wild-type mice injected with the low dose of LPS showed a peak
elevation in plasma IL-6-like activity at 1 h postinjection compared
with their saline-injected controls (Fig.
4A; P < 0.05). Although the plasma IL-6-like activity of the LPS-injected
wild-type mice began to decrease by 4 h, it was still significantly
elevated above the level of their saline-injected controls
(P < 0.05). By 24 h, the plasma IL-6
levels of the LPS-injected wild-type mice did not differ from their
saline-injected controls. IL-10 knockout mice injected with LPS showed
a virtually identical rise of plasma IL-6-like activity as the
wild-type mice at 1 h. However, at 4 h the LPS-injected knockout
mice maintained a high level of plasma IL-6-like activity that was
significantly elevated above that of their LPS-injected wild-type
counterparts (ANOVA, Fisher's P < 0.05). That is, the lack of IL-10 in the knockout mice resulted in a
sustained elevation of plasma IL-6-like activity at 4 h. By 24 h,
LPS-injected wild-type and knockout mice had virtually identical plasma
IL-6-like activity.
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Figure 4B shows the plasma
TNF--like activity in response to the low dose of LPS injected into
wild-type and IL-10 knockout mice. Injection of sterile saline did not
induce a significant elevation in plasma TNF--like activity in
either wild-type or IL-10 knockout mice at any time point following
injection. In both wild-type and IL-10 knockout mice, injection of a
low dose of LPS led to a significant rise in plasma TNF--like
activity at 1 h postinjection compared with saline-injected controls
(P < 0.05). However, the increase in
circulating TNF- did not differ between the LPS-injected wild-type
and IL-10 knockout mice at 1 h. At 4 and 24 h, plasma TNF--like
activity was no longer detectable in the LPS-injected wild-type and
IL-10 knockout mice, and there were no statistically significant
differences between groups.
Fever in response to a high dose of LPS in IL-10 knockout mice. Figure 5 shows the Tb response of wild-type and IL-10 knockout mice intraperitoneally injected with the same high, septiclike dose of LPS (2.5 mg/kg) that was injected into SW mice shown in Fig. 2. Before injection at 0 h, all groups had virtually identical Tb. The stress-induced rise in Tb at 0 and 24 h was in response to weighing and injection procedures. Due to the increased sensitivity of the IL-10 knockout mice to this dose of LPS, several of the knockout mice did not survive through the first day following injection. Therefore, only the Tb of the survivors of the 48-h observation period are presented (wild type = 100%, IL-10 knockout = 30%; data not shown). Wild-type and IL-10 knockout mice injected with sterile saline showed the normal circadian rhythm in Tb with low daytime and high nighttime values, with virtually no difference between groups. Wild-type mice intraperitoneally injected with the high dose of LPS developed an ~0.6°C fever that was significantly elevated above their saline-injected controls from 17 to 31 h (P < 0.05). Interestingly, wild-type mice developed a different Tb profile of fever to this high dose of LPS compared with the SW mice shown in Fig. 2. IL-10 knockout mice intraperitoneally injected with the high dose of LPS did not develop a fever at any time point. Starting at 10 h, knockout mice developed a profound hypothermia compared with their LPS-injected wild-type controls that was maintained through 41 h (P < 0.05).
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Changes in body weight and food intake in response to a high dose of LPS in IL-10 knockout mice. Figure 6 depicts changes in body weight and food intake of wild-type and IL-10 knockout mice injected with a high dose of LPS (2.5 mg/kg ip) or sterile saline (equivalent volume). Only those IL-10 knockout mice that survived the entire 4-day weighing period are shown. Wild-type and IL-10 knockout mice injected with sterile saline responded with virtually no change in body weight (Fig. 6A) and food intake (Fig. 6B) over 4 days following injection. Differences between saline-injected groups were not detected. The intraperitoneal injection of the high, septiclike dose of LPS induced a significant reduction in body weight and food intake in wild-type mice on day 1 compared with their saline-injected controls (P < 0.05). Body weight gradually recovered through day 4 and was paralleled by a more rapid recovery of food intake compared with their saline-injected controls. IL-10 knockout mice injected with a high dose of LPS showed a similar reduction in body weight and food intake compared with their LPS-injected wild-type counterparts on day 1. However, IL-10 knockout mice maintained a significantly reduced body weight and food intake compared with their LPS-injected wild-type controls from days 2-4 and days 2-3, respectively (P < 0.05).
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Fever in response to turpentine in IL-10 knockout mice. To induce a local inflammation, wild-type and IL-10 knockout mice were subcutaneously injected with turpentine (100 µl/mouse) into the left hindlimb. Control mice received a subcutaneous injection of sterile saline (equivalent volume) that did not alter the normal circadian rhythm of Tb (Fig. 7). Before injection, all groups of mice had virtually identical Tb. The peak in Tb immediately following injection at 0 h is a stress-induced rise due to the pain of the injection procedure. Wild-type and IL-10 knockout mice injected with turpentine responded with an ~1.5°C fever that did not differ between groups at any time point. This fever represented an ~1.0°C difference in Tb compared with their saline-injected controls. This fever peaked at ~16 h, at which time Tb began to defervesce. Wild-type and IL-10 knockout mice injected with turpentine resumed their normal circadian rhythm in Tb the second night after injection, at which time all four groups of mice had virtually identical Tb.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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We used two different murine models to examine the role of IL-10 in the Tb response to peripherally injected LPS and turpentine. Pretreatment of SW mice with rmuIL-10 significantly attenuated the febrile response to the intraperitoneal injection of a low and a high, septiclike dose of LPS. Furthermore, the hypothermia induced by a high dose of LPS was attenuated by IL-10 pretreatment. These results support the hypothesis that, pharmacologically, IL-10 can antagonize LPS-induced hypothermia and fever in mice. To test the endogenous actions of this cytokine, we examined the Tb response of IL-10 knockout mice to the peripheral injection of a low and a high, septiclike dose of LPS. These mice lack a functional gene for IL-10 in all tissues of the body and have never been exposed to the endogenous actions of this cytokine during development. IL-10 knockout mice injected with a low dose of LPS developed an exacerbated and prolonged febrile response compared with their LPS-injected C57BL/10 (wild-type) counterparts. These results suggest that endogenous IL-10 functions as an antipyretic during LPS-induced fevers in mice. On the other hand, IL-10 knockout mice showed an enhanced sensitivity to the injection of a high, septiclike dose of LPS in terms of their Tb, body weight, and food intake responses. Whereas wild-type mice developed a fever 24 h after a high dose of LPS, IL-10 knockout mice either succumbed to the infection (30% survival in IL-10 knockout mice vs. 100% survival in wild-type mice) or developed a prolonged hypothermia. In addition, in response to the high dose of LPS, IL-10 knockout mice showed sustained reductions in body weight and food intake compared with their wild-type counterparts. These results confirm earlier findings of a protective role of IL-10 in septic shock (8, 10, 36).
Release of IL-10 in response to LPS has been reported. For example, sepsis induced by CLP or LPS injection induced elevated plasma levels of IL-10 in mice (36). Other studies have demonstrated IL-10 production from human hypothalamus and pituitary, supporting a role in bidirectional communication between the neuroendocrine and immune systems (31). Recently, two studies showed a role for IL-10 in the febrile response to endotoxin injection. Rats treated intracerebroventricularly with IL-10 developed attenuated fevers to the peripheral injection of LPS (27). These data support a role for central IL-10 in the regulation of fever during systemic inflammation induced by a bacterial stimulus. Opp et al. (29) similarly showed central inhibitory effects of muIL-10 on spontaneous sleep in rats. Many of the cytokines implicated in the regulation of sleep have also been shown to regulate fever (29). An antipyretic action of IL-10 on LPS fever in human volunteers has also been reported (30).
IL-10 pretreatment, but not posttreatment, has been shown to attenuate
fever and cytokine release in response to endotoxin injection in humans
(30). Howard et al. (10) reported protection of mice from the lethality
of endotoxemia only when mice were treated no later than 30 min after
the LPS injection. In the present study, we chose to inject IL-10 into
SW mice immediately before the injection of LPS. We observed an
immediate (~1-3 h) inhibition of hypothermia and fever in
response to the different doses of LPS. Although we did not measure
plasma cytokine levels in those mice, it is presumed that the
antagonism of LPS effects by IL-10 was due to reduction in the plasma
concentration of IL-6 or TNF-. Several studies have reported similar
time courses of effects of IL-10 on cytokine release in vivo (30, 35).
We have shown previously in our laboratory that the hypothermic effect
of LPS in mice is regulated by endogenous TNF-. SW mice pretreated
with the soluble TNF receptor or TNF antiserum developed an attenuated
hypothermia in response to the same high, septiclike dose of LPS used
in the present study (15). Similarly, in response to sepsis induced by
CLP, TNF p55/p75 receptor knockout mice developed an attenuated
hypothermia compared with their wild-type controls, whereas fever was
not affected (22). Although injection of pharmacological doses of
TNF- results in fever in several species, there is little evidence
that TNF- mediates fever in mice. On the other hand, IL-6 is thought
to be a key mediator of LPS fevers. Chai et al. (3) found that
IL-6-deficient mice failed to develop fever to the intraperitoneal
injection of a low dose of LPS (50 µg/kg ip). In our laboratory, IL-6
knockout mice injected with the same high, septiclike dose of LPS used in the present study developed normal fevers (16). Together, these data
indicate that the role of these cytokines in the febrile response may
differ depending on the injected dose of LPS. In the present study, we
were unable to assess plasma levels of IL-6 and TNF- in the SW mice
due to a lack of availability of rmuIL-10.
In response to a low dose of LPS, IL-10 knockout mice developed an
exacerbated and prolonged fever compared with their wild-type controls.
To examine the mechanism(s) responsible for the exacerbated fevers in
the knockout mice, we measured plasma levels of IL-6- and TNF--like
activity at several time points following injection. Our results show a
correlation between a sustained elevation in plasma IL-6 levels at 4 h
postinjection in the IL-10 knockout mice and their early (i.e.,
4-11 h) exacerbated febrile response. On the other hand, the late
(i.e., 20-28 h) febrile response in the knockout mice did not
correlate with an alteration in the plasma level of IL-6 or TNF-.
Interestingly, plasma TNF--like activity did not differ in the
wild-type and knockout mice at any time point following LPS. These
results correlate with our earlier findings of an inability to detect
differences in the Tb responses of
wild-type and TNF p55/p75 receptor knockout mice to a low dose of LPS
(21). Thus TNF- does not appear to mediate fever to a low dose of
LPS in mice, and other putative mediators of fever may be responsible
for the altered febrile response of the knockout mice in the present
study. The mechanism responsible for the late fever in the IL-10
knockout mice was not elucidated in this study. We have injected this
same low dose of LPS (50 µg/kg ip) in several types of wild-type and
knockout mice in our laboratory and never detected a fever 24 h after
injection, indicating an enhanced sensitivity of the IL-10 knockout
mice to this dose. Similar data were reported in a study by Berg et al.
(2) in which lethality was induced at a 40-fold lower dose of LPS in IL-10 knockout compared with wild-type mice. Enhanced plasma levels of
TNF-, IL-12, IL-1, and IFN- were detected in the IL-10
knockout mice by Berg et al. (2). The production of IL-12 during septic shock appears to be partially responsible for tissue injury and death
(37). In light of these reports, we must consider the possibility that
alterations in the plasma levels of one or more of these cytokines, or
others perhaps not yet identified, may be responsible for the altered
fevers and other acute phase responses in the IL-10 knockout mice (or
in the SW mice in response to IL-10 pretreatment) in response to LPS.
An additional putative mechanism of IL-10 antipyretic action could be through the release of endogenous glucocorticoids, or other putative endogenous antipyretics. IL-10 has been shown to stimulate secretion of glucocorticoids in humans (31). Glucocorticoids have been shown to inhibit the in vitro and in vivo production of IL-6 (26, 34). In addition, several studies have demonstrated exacerbated fevers in rats exposed to LPS or psychological stress following treatment with the glucocorticoid II receptor antagonist RU-38486 (25, 26). The exacerbated fevers to LPS correlated with increased plasma concentration of IL-6 (26). These effects of glucocorticoids on LPS-induced fevers have also been shown to be centrally mediated in the rat (25). Because we did not measure the level of glucocorticoids in our murine model, we are unable to assess the contribution of this steroid on the observed changes in the febrile response to LPS in our knockout mice.
In response to the high, septiclike dose of LPS, IL-10 knockout mice
showed a virtually identical reduction in body weight and food intake
as their wild-type controls on day 1.
Whereas LPS-treated wild-type mice began to recover their normal body weight and food intake by day 2, IL-10
knockout mice showed a sustained reduction in body weight and a more
gradual recovery of food intake through day
4. Previous studies in our laboratory have been unable
to detect dramatic differences in the body weight and food intake
responses of wild-type and knockout mice (e.g., IL-1, IL-6, TNF
p55/p75 receptor knockout mice) to the injection of LPS or sepsis
induced by CLP (17, 21, 22). Despite evidence in the literature for a
role of these cytokines in the regulation of body weight and food
intake during infection, our previous data suggested the development of
redundancies in the control of these variables in many types of
knockout mice. Because we only measured survival through
night 5 post-LPS, it is unclear whether the dramatic differences detected were an indication of eventual lethality in these mice or simply a dysregulation of these
variables due to the lack of endogenous IL-10 control.
IL-10 knockout mice showed a prolonged hypothermia and enhanced
lethality in response to the high dose of LPS. Van der Poll et al. (35,
36) have shown a protective role of IL-10 for mice during sepsis
induced by CLP, a finding that correlated with inhibition of plasma
concentrations of TNF- (35, 36). Surprisingly, anti-TNF treatment of
the mice did not provide protection from the lethality of CLP, perhaps
due to methodological complications related to the timing of anti-TNF
treatment. Because we have shown that TNF p55/p75 receptor knockout
mice are protected from the hypothermia and lethality of sepsis induced
by CLP (22), it is reasonable to hypothesize that the lethality we
observed in the IL-10 knockout mice was due to enhanced plasma levels
of TNF-. This would correlate with the enhanced hypothermia observed
in these mice as well (see discussion above). Unfortunately, the rapid
lethality of sepsis prevented us from measuring plasma cytokine levels
in the IL-10 knockout mice.
In response to the local inflammatory stimulus turpentine, wild-type
and IL-10 knockout mice developed virtually identical fevers. These
data suggest that either 1)
endogenous IL-10 is not involved in the febrile response or
2) redundancies have developed in
the regulation of fevers to turpentine in the knockout mice. To the
best of our knowledge, the role of endogenous IL-10 in the
Tb response to turpentine in mice
has not been investigated. We hypothesized that IL-10 knockout mice
would develop an exacerbated febrile response to turpentine on the
basis of the reported ability of IL-10 to inhibit the production of
several cytokines, including IL-1 and IL-6. Although TNF-
production is also inhibited by IL-10 and has been implicated in fevers
to turpentine in rats, there are currently no data to support the
hypothesis that TNF- regulates turpentine fevers in mice. In fact,
we have shown virtually identical fevers in wild-type and TNF p55/p75
receptor knockout mice injected subcutaneously with turpentine (21).
However, we cannot eliminate the possibility that TNF- is involved
in turpentine fevers in wild-type mice while absence of its action is
effectively compensated for in the TNF p55/p75 receptor knockout mice.
In any case, TNF- appears to be an important regulator of fevers to
a systemic inflammation induced by LPS, but not to a local inflammation
in response to turpentine in mice. On the other hand, IL-1 and IL-6
are critical mediators of the febrile response to turpentine. Mice
deficient in IL-1 (38), the IL-1 type I receptor (the only known
signaling receptor for IL-1; Ref. 20), or IL-6 (16) are resistant
to fevers, body weight loss, and anorexia induced by the same dose of
turpentine used in the present study. These data are similar to those
reported by Oldenburg et al. (28) and Gershenwald et al. (9)
demonstrating the efficacy of antibody against IL-6 or the IL-1 type I
receptor, respectively, in reducing several of the acute phase
responses to turpentine in mice. Although we were unable to detect any
differences in the febrile response to turpentine in the wild-type and
IL-10 knockout mice, this again may simply be a reflection of redundant cytokine actions in vivo in the mediation of this response. Over the
past several years our laboratory has been actively involved in
experimentation with several types of knockout mice for the study of
thermoregulatory mechanisms in response to inflammation. Certainly, the
most significant contribution of this model to the study of fever and
sickness behaviors is the ability to study the effect of the
elimination of a cytokine's action from all tissues of the body.
However, the caveat to this approach is that these mice have never been
exposed to the cytokine's action during their development. Therefore,
it is informative to use more traditional pharmacological approaches,
such as antibody administration, to confirm negative results generated
with the use of gene knockout mice.
| |
ACKNOWLEDGEMENTS |
|---|
Research supported by National Institute of Allergy and Infectious Diseases Grant AI-27556.
| |
FOOTNOTES |
|---|
Present address and address for reprint requests: L. R. Leon, Diabetes Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bldg. 10, Rm. 8N-252, 10 Center Drive MSC 1770, Bethesda, MD 20892-1770.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Received 24 March 1998; accepted in final form 10 September 1998.
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REFERENCES |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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