Am J Physiol Regulatory Integrative Comp Physiol 282: R702–R709, 2002;
10.1152/ajpregu.00393.2001.
Effect of interleukin-18 on mouse core body temperature
SILVIA GATTI,1 JENNIFER BECK,1 GIAMILA FANTUZZI,2
TAMAS BARTFAI,1 AND CHARLES A. DINARELLO2
1
Pharmaceutical Division, CNS Preclinical Research, F. Hoffmann-LaRoche,
CH-4070 Basel, Switzerland; and 2Division of Infectious Diseases,
University of Colorado Health Science Center, Denver, Colorado 80262
Received 9 July 2001; accepted in final form 11 October 2001
INTERLEUKIN-18 (IL-18), initially characterized as interferon-␥ (IFN-␥)-inducing factor, is mainly produced by
activated macrophages and participates in the T helper
cell type 1 (Th1) response during immunorecognition
(33). IL-18 acts as a cofactor, inducing the production of
IFN-␥ usually in the presence of another stimulus [for
instance, lipopolysaccharide (LPS) or IL-12], and potentiates Th1- and natural killer cell-induced cytotoxicity by increasing the expression of Fas ligand (6, 33).
The proinflammatory role of IL-18 has been extensively characterized in vitro, showing that IL-18 promotes the innate immune response, mainly inducing
the production of tumor necrosis factor (TNF)-␣, IL1␣/␤, IL-6, IL-8, macrophage inflammatory protein-1␣,
and monocyte-chemoattractant protein-1 (12, 29, 34,
37) with no direct effect on the production of prostaglandins (PGs; Ref. 38). In vitro studies have also
confirmed that mature IL-18 is produced by various
cell types in response to endotoxins (LPS), and in vivo
studies showed that IL-18 could be partially responsible for the LPS-induced lethality in mice (20, 39).
During sepsis, circulating IL-18 levels are increased in
humans (16).
The similarities between IL-18 and IL-1␣/␤, both in
structural and in functional terms, have been highlighted by several studies. Both IL-1 and IL-18 gene
expression are induced by LPS. However, differently
from all other proinflammatory cytokines, 1) IL-18
gene expression is constitutively high, 2) IL-18 mRNA
and IL-18 precursor protein (24 kDa) are broadly expressed in several tissues and cell types (4, 25, 30, 35,
36), and 3) IL-18 actions may be regulated by changes
in concentration of IL-18 binding protein. Neuroendocrine cells in adrenal glands and pancreas (21) also
express pro-IL-18 mRNA, possibly in response to stress
events. By RT-PCR it was possible to demonstrate the
constitutive expression of the mature transcript coding
for IL-18 precursor in rat brain (42), probably mainly
in astrocytes and microglia (4).
Similarly to IL-1␤ precursor, the pro-IL-18 is cleaved
to the mature active form mainly by IL-1␤-converting
enzyme, and in the case of IL-18 the enzymatic cleavage is probably the main mechanism of control on the
production of bioactive IL-18 (11, 14).
The cellular effects of IL-18 are a consequence of the
specific interaction with the membrane-bound IL-18
receptor (IL-18R) complex (IL-18R␣/␤). Signal transduction by IL-18 is highly analogous to that observed
in the case of IL-1R type I (IL-1RI) (33). After binding
to the IL-18R␣ chain (24), IL-18/IL-18R␣ complex recruits the IL-18 accessory protein (IL-18R␤) (5, 23).
This increases the affinity of the ligand for the receptor
complex, and a signal is transduced. A soluble IL-18binding protein binds and neutralizes IL-18 effects,
reducing the amount of cytokine available for the interaction with IL-18R complex (38). In the periphery
Address for reprint requests and other correspondence: S. Gatti,
Hoffmann-LaRoche PRBN-P, Bldg.68, Rm.40B, CNS Preclinical Research, Pharmaceutical Division, Grenzacherstrasse, CH-4070 Basel,
Switzerland (E-mail: [email protected]).
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. Section 1734
solely to indicate this fact.
interleukin-1␤; interferon-␥
R702
0363-6119/02 $5.00 Copyright © 2002 the American Physiological Society
http://www.ajpregu.org
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.1 on June 17, 2017
Gatti, Silvia, Jennifer Beck, Giamila Fantuzzi, Tamas Bartfai, and Charles A. Dinarello. Effect of interleukin-18 on mouse core body temperature. Am J Physiol Regulatory Integrative Comp Physiol 282: R702–R709, 2002;
10.1152/ajpregu.00393.2001.—We have studied, using a telemetry system, the pyrogenic properties of recombinant murine interleukin-18 (rmIL-18) injected into the peritoneum of
C57BL/6 mice. The effect of IL-18 was compared with the
febrile response induced by human IL-1␤, lipopolysaccharide
(LPS), and recombinant murine interferon-␥ (rmIFN-␥). Both
IL-1␤ and LPS induced a febrile response within the first
hour after the intraperitoneal injection, whereas rmIL-18
(10–200 ␮g/kg) and rmIFN-␥ (10–150 ␮g/kg) did not cause
significant changes in the core body temperature of mice.
Surprisingly, increasing doses of IL-18, injected intraperitoneally 30 min before IL-1␤, significantly reduced the IL-1␤induced fever response. In contrast, the same pretreatment
with IL-18 did not modify the febrile response induced by
LPS. IFN-␥ does not seem to play a role in the IL-18-mediated attenuation of IL-1␤-induced fever. In fact, there was no
elevation of IFN-␥ in the serum of mice treated with IL-18,
and a pretreatment with IFN-␥ did not modify the fever
response induced by IL-1␤. We conclude that IL-18 is not
pyrogenic when injected intraperitoneally in C57BL/6 mice.
Furthermore, a pretreatment with IL-18, 30 min before IL1␤, attenuates the febrile response induced by IL-1␤.
R703
IL-18 AND FEVER
MATERIALS AND METHODS
Mice. C57BL/6J male mice (25–30 g, Biological Research
Laboratories, Füllinsdorf) were used for this study. They
were individually housed with free access to food and water
at vivarium temperature of 25°C (30–40% humidity) for 7–10
days before the surgery (light from 7 AM to 7 PM). The
animals were acclimated at 29.5°C after the surgery. All
animals were kept on the same standard diet, Kliba no. 243
(12.6 MJ/kg).
Treatments. LPS was from Escherichia coli (serotype 026:
B6, Sigma lot 107H4091, cell culture tested). Recombinant
human (rh) IL-1␤ was from R&D Systems (Minneapolis, MN;
helper T cells proliferation assays: ED50 ⫽ 5–10 pg/ml).
Recombinant murine (rm) IFN-␥ was from R&D Systems
(antiviral test in L929; ED50 ⫽ 0.1–0.4 ng/ml). rmIL-18 was
expressed and purified by Peprotech (Rocky Hill, NJ) (23).
rmIL-12 was a kind gift of Genetics Institute (Andover, MA);
the specific activity of IL-12 was 2.7 ⫻ 106 U/mg. LPS and
cytokines were reconstituted in pyrogen-free saline solution.
Aliquots were stored at ⫺20°C, and each frozen aliquot was
used only once. LPS or cytokines were injected intraperitoneally in pyrogen-free saline solution. Control mice were
injected with pyrogen-free saline solution. All treatments
were carried out between 9 AM and 11 AM. The volume of
injection was calculated in proportion to the body weight, and
it ranged between 0.1 and 0.2 ml. The total volume of liquids
injected into the peritoneum, during multiple treatments,
never exceeded 0.4 ml.
Implant of telemetry probes. Telemetry probes (Vitalview
4000, MiniMitter, Sunriver, OR) were inserted into the
mouse peritoneum under systemic anesthesia with diazepam
(5 mg/kg ip) and ketamine (100 mg/kg ip). For the postoperative analgesic treatment, buprenorphine (0.05 mg/kg sc)
was used twice a day for 1 day after the surgery. Animals
were then acclimated for 5–7 days at 29.5 ⫾ 0.5°C of ambient
temperature (30–40% humidity) before any further treatment.
Measurement of core body temperature in mice. Core body
temperature and horizontal motor activity values were recorded every minute in freely moving animals housed in
single cages, starting from the evening before the day of the
experiment and until 18 h after the injections. This study
received the approval of the Cantonal Veterinary office of the
city of Basel, Switzerland.
Measurement of IFN-␥ levels in the serum of mice treated
with IL-18. Mice (8 wk old) were treated for 4 days with IL-18
or murine IL-12 (400 ng/mouse ip). The serum was taken
from the retroorbital plexus 2 h after the last injection. IFN-␥
levels were measured with an enzyme-linked immunosorbent
assay kit from Endogen (Wobur, MA).
Statistical analysis. Core body temperature was recorded
every minute, and data were averaged every 10 min. The
results were analyzed using ANOVA followed by post hoc
tests (UNISTAT software package).
RESULTS
Effect of IL-18, IL-1␤, and LPS on core body temperature of C57BL/6 mice. IL-1␤ (10 ␮g/kg ip) causes fever
in mice acclimated in thermoneutral environment
(29.5 ⫾ 0.5°C) (Fig. 1A). During the fever response, the
core body temperature increases about 1–1.5°C, with a
maximal rise within 2 h after the intraperitoneal injection. The core body temperature of mice injected
with IL-1␤ is different from that of saline-injected
animals for ⬃6 h after the treatment. The core body
temperature of treated mice was not significantly different from that of mice injected with saline solution
⬃24 h after the treatment (data not shown).
Under the same experimental conditions, LPS (50
␮g/kg ip) triggers a similar febrile response within 1 h
after the injection, and the fever reaches peak elevation within the second hour after the injection (data not
shown). The LPS used during this study is highly
purified cell culture-tested LPS from E.coli with ⬍1%
protein content. The doses of the two pyrogens IL-1␤
and LPS were chosen as the minimal doses causing a
reproducible febrile response in this strain of mice
(data not shown).
Animals consistently exhibited a similar stress reaction immediately after the intraperitoneal injection,
with a sudden hyperthermia lasting ⬃45 min after the
injection (Fig. 1). This reaction was longer (⬃60 min) in
the case of animals injected twice (Fig. 2).
Under our experimental conditions, neither rmIL-18
(10–50 ␮g/kg) nor rmIFN-␥ (10–150 ␮g/kg) (Fig. 1, B
and C, respectively) induced a sustained increase of the
core body temperature within 5–6 h after the treatment. No changes in the circadian rhythm of the core
AJP-Regulatory Integrative Comp Physiol • VOL
282 • MARCH 2002 •
www.ajpregu.org
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.1 on June 17, 2017
the distribution of IL-18R␣ is rather broad, and IL18R␣ mRNA can be detected mainly in lymphocytes
and hematopoietic cells (32).
The intracellular, postsignal cascade of IL-18R kinases is nearly identical to that of IL-1RI. 1) IL-1RI
and IL-18R signaling involves the recruitment of
MyD88 and the activation of a common kinase, IL-1
receptor-associated kinase (IRAK; Refs. 17, 22). 2) Another kinase, known as TNF receptor (TNFR)-activating factor-6 (TRAF-6), contributes to the signal transduction system of both IL-1 and IL-18 receptors (27)
and results in activation of nuclear factor (NF)-␬B (26,
41). NF-␬B translocation to the nucleus is associated
with initiation of cyclooxygenase-2 (COX-2) gene expression.
The observed functional similarities between IL-18
and IL-1␣/␤ suggest the presence of partially overlapping roles for these cytokines in the control of the acute
phase response during infection. This is also suggested
by the recent study of Kubota et al. (28), showing that
IL-18 promotes sleep in rabbits and rats.
Considering that IL-1␤ is the main endogenous pyrogen and that IL-1␤-induced fever is mediated by
intrahypothalamic production of PGE2 (8), we studied
the pyrogenic properties of IL-18 and compared the
effect of IL-18 with the febrile response triggered by
IL-1␤ and LPS, respectively.
IL-18 is not inducing PGE2 production in peripheral
cells; this observation, however, is not per se predictive
of a lack of pyrogenicity of this proinflammatory cytokine. IL-6, an endogenous pyrogen in humans and
rabbits, does not induce COX-2 expression and PG
production in monocytes or synovial fibroblasts. Moreover, IFN-␥, which is also pyrogenic in humans, suppresses LPS- and IL-1-induced PG production in monocytes. Therefore, to resolve this issue, we have tested
the pyrogenicity of recombinant murine IL-18 in mice.
R704
IL-18 AND FEVER
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.1 on June 17, 2017
Fig. 1. Effect of intraperitoneal injection of cytokines on core body temperature of C57BL/6 mice.
A: effect of interleukin (IL)-1␤ (10 ␮g/kg ip, n ⫽
5) compared with saline-injected controls (n ⫽ 6).
Core body temperature was recorded every
minute, and data were averaged every 10 min.
One hour of basal recording before the treatment
is shown. Data are means ⫺ SE. * P ⬍ 0.01, IL-1␤
vs. saline treatment (1-way ANOVA followed by
Dunnett’s test). B: effect of IL-18 (10 ␮g/kg ip,
n ⫽ 8; 50 ␮g/kg ip, n ⫽ 2) compared with salineinjected mice (n ⫽ 6). Data are means ⫺ SE. C:
effect of interferon (IFN)-␥ (10 ␮g/kg ip, n ⫽ 2; 50
␮g/kg ip, n ⫽ 2; 150 ␮g/kg ip, n ⫽ 2) compared
with saline-injected control group (n ⫽ 6).
body temperature were observed during the 24 h after
the treatment (data not shown).
No signs of sickness or changes in horizontal locomotor activity were observed in mice treated with IL-18.
The doses of rmIL-18 used in this study (10–50
␮g/kg; 0.25–1.25 ␮g/mouse) are in the range of IL-18
doses active in suppressing the growth of MetA tumor
cell ascites and increasing histidine decarboxylase activity in mouse tissues (31, 43).
At a dose of 200 ␮g/kg ip of IL-18, no sustained effect
on core body temperature was observed (data not
shown).
Effect of a pretreatment with IL-18 or IFN-␥ on IL1␤- and LPS-induced fever. As described above, mice
pretreated (30 min) with IL-18 (10–200 ␮g/kg ip) developed a reduced febrile response to IL-1␤ (10 ␮g/kg
ip) (Fig. 2). This reduction in IL-1␤-induced fever by
IL-18 was dose dependent. In fact, the pretreatment
with IL-18 at 10 ␮g/kg resulted in a slight reduction of
IL-1␤-induced fever, whereas the injection of 50 ␮g/kg
of IL-18 30 min before IL-1␤ resulted in a significant
shortening of the febrile period in each of the IL-18treated animals. The highest IL-18 dose tested in this
study, 200 ␮g/kg, completely blocked IL-1␤-induced
AJP-Regulatory Integrative Comp Physiol • VOL
282 • MARCH 2002 •
www.ajpregu.org
R705
IL-18 AND FEVER
Fig. 2. Effect of a pretreatment
with increasing doses of IL-18 on
the febrile response induced by
IL-1␤ in mice. IL-18 (10 ␮g/kg, n ⫽
8; 50 ␮g/kg, n ⫽ 5; 200 ␮g/kg, n ⫽ 2)
was injected 30 min before the injection of IL-1␤ (10 ␮g/kg ip). Control animals were injected with saline solution 30 min before IL-1␤
injection (n ⫽ 7) or saline injection
(n ⫽ 10). Data are means ⫺ SE.
** P ⬍ 0.05, IL-18 (50 ␮g/kg) plus
IL-1␤ vs. saline plus IL-1␤ (ANOVA
followed by Student-NewmanKeuls test).
DISCUSSION
Fever is a stereotypic response to endogenous and
exogenous pyrogens, associated with increased serum
levels of proinflammatory cytokines (IL-1␣/␤, TNF-␣,
and IL-6) and hypothalamic production of PGs (mainly
PGE2), that trigger fever interacting with EP3 receptors (13). Exogenous pyrogens, like LPS, trigger fever
via the interaction with Toll-like receptors (TLR) expressed in macrophagic cells, endothelial cells, or
perivascular cells of the organum vasculosum lamina
terminalis, with either direct release of PGE2 or the
subsequent release of endogenous pyrogens (i.e., cytokines) (13).
In the case of intraperitoneal injection of IL-1␤ (10
␮g/kg) into mice, the febrile response is only partially
due to the circulating proinflammatory cytokine (18).
In fact, the effect of the intraperitoneal injection of
IL-1␤ is amplified by the local production of IL-1␣/␤,
TNF-␣, and IL-6 by peritoneal macrophages, and fever
is also triggered via the activation of the sensory afferent part of the vagal nerve. In rats a surgical cut of the
sensory part of this nerve in the subdiaphragmatic
region completely prevents the development of IL-1␤induced fever after intraperitoneal injection of IL-1␤
Fig. 3. Effect of a pretreatment with
IL-18 on LPS-induced fever in mice.
IL-18 (50 ␮g/kg, n ⫽ 3) was injected 30
min before LPS-induced fever (50
␮g/kg ip, n ⫽ 3).
AJP-Regulatory Integrative Comp Physiol • VOL
282 • MARCH 2002 •
www.ajpregu.org
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.1 on June 17, 2017
fever response. In contrast, similar attenuation of LPSinduced fever (50 ␮g/kg) was not observed with a pretreatment by IL-18 (50 ␮g/kg) (Fig. 3). Thus, in our
experimental conditions, the suppression of fever by
pretreatment with IL-18 (50 ␮g/kg ip) appears to be
effective for IL-1␤ and not for LPS.
The time interval of pretreatment seems to be crucial
for the IL-18 effect in suppressing IL-1␤ fever in mice.
In fact, mice pretreated with the highest dose of IL-18
used in this study (200 ␮g/kg) mounted a normal IL1␤-induced febrile response when IL-18 was injected
1 h before IL-1␤ (Fig. 4) compared with 30-min pretreatment (Fig. 2).
Because IL-18 is an inducer of IFN-␥, particularly
with IL-12 or other T-cell activators, we examined the
possible effect of IFN-␥ on IL-1␤-induced fever. A pretreatment (30 min) with IFN-␥ (50 ␮g/kg ip) did not
reduce IL-1␤-induced fever under these experimental
conditions (Fig. 5).
Serum levels of IFN-␥. IFN-␥ was increased slightly
in the circulation of mice injected each day, for 4 days,
with IL-18 (16 ␮g/kg ip). However, the serum levels
were not statistically different from those observed in
control mice injected with saline (Table 1). In contrast,
mice injected with IL-12 exhibited high levels of IFN-␥.
R706
IL-18 AND FEVER
Fig. 4. Effect of a high dose of IL-18 on IL-1␤-induced
fever in mice when IL-18 is injected 1 h before IL-1␤.
IL-18 (200 ␮g/kg, n ⫽ 3) was injected 1 h before IL-1␤
(10 ␮g/kg) intraperitoneally. Control animals were
injected with saline solution 1 h before IL-1␤ injection
(n ⫽ 2) or saline injection (n ⫽ 2).
was sustained in our experimental conditions by the
induction of several endogenous pyrogens (TNF-␣, for
instance). In fact, LPS-induced fever is still present in
IL-1␣/␤ knockout. Moreover, LPS-induced fever, in
rabbits and in humans, is not affected by the coinfusion
of IL-1 receptor antagonist (IL-1ra; Ref. 13).
Human IFN-␥ causes fever when injected subcutaneously in humans or intraperitoneally in rabbits. In
mice, however, rmIFN-␥ does not cause fever when
injected in doses ranging from 10 to 150 ␮g/kg ip (2).
In the same experimental model, murine IL-18 (10–
200 ␮g/kg ip) does not cause a significant and sustained increase of the core body temperature (Fig. 1).
These results confirmed our earlier unpublished observations using human IL-18 in mice.
Recently, Reznikov et al. (38) reported a significant
biological difference between the two proinflammatory
cytokines: IL-1␤ and IL-18. In vitro, IL-18 does not
trigger PGE2 production in human peripheral blood
mononuclear cells and, when coincubated with IL-1␤,
even causes a marked reduction of PGE2 production
(IFN-␥ mediated). Therefore, in vivo studies are required to understand if IL-18 is not causing fever
because of a lack of production of PGE2 in the hypothalamus. However, the evidence that IL-18 is not
pyrogenic when injected intraperitoneally in mice sug-
Fig. 5. Effect of a pretreatment
with IFN-␥ on IL-1␤-induced fever
in mice. Recombinant murine
IFN-␥ (50 ␮g/kg ip, n ⫽ 3) was injected 30 min before IL-1␤ (10
␮g/kg ip). Control animals were injected with saline solution 30 min
before IL-1␤ injection (n ⫽ 7) or
saline injection (n ⫽ 10).
AJP-Regulatory Integrative Comp Physiol • VOL
282 • MARCH 2002 •
www.ajpregu.org
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.1 on June 17, 2017
(19). Moreover, the synthesis of IL-1␤ can be detected
in the vagal nerve and in the nodose ganglion soon
after the intraperitoneal injection of LPS (15). Therefore, we hypothesized that intraperitoneal injection of
IL-18 would also cause fever by similar mechanisms.
However, intraperitoneal injection of IL-18 did not
produce fever.
Endogenous or exogenous pyrogens can cause fever
in all endotherms, because they trigger the shift upward of the hypothalamic core temperature set point,
with a subsequent increase in metabolic rate and facultative thermogenesis. In the case of small rodents
like mice and rats, it is necessary to acclimate the
animals in thermoneutral conditions (29–30°C of ambient temperature for mice) before the treatment, to
allow them to mount a proper febrile response (16).
C57BL/6 mice are an inbred strain commonly used
for studies of in vivo effects of cytokines and of T-cellmediated immune responses associated with the production of IFN-␥ (1, 3).
In the experimental conditions used in the present
study both IL-1␤ (10 ␮g/kg ip) and LPS (50 ␮g/kg ip)
cause a prolonged fever response in C57BL/6 mice.
LPS- and IL-1␤-induced fevers in mice are very similar
both in terms of time course and of magnitude of the
effect (data not shown) despite the fact that LPS fever
R707
IL-18 AND FEVER
Table 1. IFN-␥ levels in the serum of mice treated
with IL-18 and IL-12
Treatments
IFN-␥, ng/ml
Saline
IL-18
IL-12
0.14 ⫾ 0.07
0.58 ⫾ 0.32
18.66 ⫾ 9.29
Data are means ⫾ SE; n ⫽ 4. Mice were treated for 4 days with
saline, interleukin (IL)-18 (16 ␮g/kg), or IL-12 (16 ␮g/kg), and the
serum was taken 2 h after the last injection. IFN-␥, interferon-␥.
Perspectives
New ligands belonging to the structural IL-1 family
have been recently discovered together with a large
series of TLR receptors. All the known ligands of the
IL-1R structural superfamily exhibit high selectivity
for the different receptors, raising a lot of interesting,
still unanswered, questions about the structural requirements of ligand-receptor interaction for agonist
activity and about the common or different intracellular complexes involved in signal transduction. The
picture is getting broader and more detailed, mostly
thanks to in vitro studies.
AJP-Regulatory Integrative Comp Physiol • VOL
282 • MARCH 2002 •
www.ajpregu.org
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.1 on June 17, 2017
gests a main difference in the neurological effects of
IL-1␤ and IL-18.
Experimental antipyretic strategies usually block
the effect of endogenous pyrogens (IL-1␤, IL-6, and
TNF-␣) via the inhibition of PGE2 formation by inhibiting COX enzymes (COX-1 and/or COX-2). In each
case, the antipyretic agent is injected 30 min before the
pyrogen. Using a classical protocol for the study of
antipyretic agents, we show in the present study that
IL-18 reduces the fever response induced by rhIL-1␤
but not LPS in a dose-dependent manner.
It is unlikely that IFN-␥ is responsible for the observed effect of IL-18 on IL-1␤ fever. In fact, rmIL-18
(16 ␮g/kg) injected intraperitoneally in mice for 4 days
does not increase IFN-␥ levels in the serum, and, more
importantly, a pretreatment with IFN-␥ (50 ␮g/kg ip)
does not modify IL-1␤-induced fever (Fig. 5).
A central production of IFN-␥ at the level of periventricular organ or hypothalamus could, however, play a
role in the control of the pyrogenic response induced by
IL-1␤. Similarly to what was observed by Reznikov et
al. (38) in blood mononuclear cells, central IL-1␤-induced PG production could be reduced by a cotreatment with IL-18 because of the local induction of
IFN-␥.
Several peripheral or central events could as well
account, at least partially, for the observed pharmacological IL-18 antagonism on IL-1␤-induced fever. IL-18
could, for instance, act on endogenous antipyretic/antiinflammatory mechanisms such as IL-4 secretion, the
epoxygenase pathway, or the central release of arginine vasopressin or IL-1ra, for instance (13).
However, the lack of effect of IL-18 pretreatment on
LPS-induced fever suggests the presence of cellular
events specific for IL-1RI-mediated signal transduction. The observed attenuation of IL-1␤ fever could be,
for instance, the result of mechanisms of sequestration
at the receptor accessory protein level or at the level of
intracellular signaling proteins, like MyD88 or IRAK.
In vitro studies are necessary to further address this
hypothesis. However, when considering this hypothesis, we should remember that LPS also mobilizes
IRAK1/2-TRAF6. Thus possible functional interactions
between IL-18 and IL-1 receptor signaling must be
upstream from IRAK1/2-TRAF6 in the TLR-signaling
pathway.
The effect of IL-18 on IL-1␤ fever is dependent on the
time protocol of the pretreatment. IL-18 (200 ␮g/kg) is
able to inhibit completely IL-1␤-induced fever when
injected 30 min before IL-1␤ (Fig. 2), whereas the effect
is no longer present if the same dose of IL-18 is injected
1 h before IL-1␤ (Fig. 4).
It is rather difficult to explain the time dependence of
the antipyretic effect of the IL-18 pretreatment with
the present, rather scanty, knowledge of in vivo IL-18
effects. Our experimental observation could be consistent with the hypothesis of cross-talk between fast
intracellular events associated with the activation of
IL-1R and/or IL-18 receptor complexes.
A similar time-dependent effect could be observed
while studying the antipyretic effect of IL-1ra pretreatment: in this case, the blockade of IL-1 receptors with
IL-1ra is effective on IL-1␤-induced fever response
when IL-1ra is injected 15 min before IL-1␤ (7, 13) or
after IL-1␤ because of the short plasma half-life of
IL-1ra. Pharmacokinetic reasons cannot be ruled out
also in the case of the observed time dependence of the
IL-18 effect.
Moreover, because of the tight time dependence of
the IL-18 effect on IL-1␤, we cannot rule out completely
that the observed lack of effect of IL-18 on LPS-induced
fever is due to the protocol of treatment we used. We
are further exploring this possibility.
This is the first study addressing the effect of a
systemic treatment with IL-18 on core body temperature. We conclude that IL-18 is not causing fever per se
when injected intraperitoneally in C57BL/6 mice.
These results are in agreement with the in vitro observation that IL-18 is not able to trigger the production of
PGE2 in macrophage-like cells (38) and with the study
of Kubota et al. (28) showing no changes in brain
temperature in rats treated with intraperitoneal IL-18.
Moreover, a recent study carried out by Stuyt RJL,
Netea MG, Kullberg BJ, and van der Meer JWM,
(unpublished data) has further confirmed that recombinant human IL-18 (1 ␮g/kg iv) is not pyrogenic in
rabbits.
IL-18 (10–200 ␮g/kg ip) exhibits an in vivo effect
when tested in a protocol of pretreatment on IL-1␤induced fever. In fact, we could observe a significant
reduction of IL-1␤-induced fever when IL-18 is injected
30 min before the pyrogen. Considering that this effect
is specific for IL-1␤-induced fever and that it is not
observed when IL-18 is injected 1 h before IL-1␤, we
suggest the presence of cross-talk at the level of early
IL-1R-mediated intracellular events.
R708
IL-18 AND FEVER
This study was supported in part by National Institutes of Health
Grant AI-15614 (to C. A. Dinarello).
Present address of T. Bartfai: “Harold L. Dorris” Neurological
Research Center, Dept. of Neuropharmacology, Scripps Research
Institute, 10550 N. Torrey Pines Rd., SR-307, La Jolla, CA 92037.
REFERENCES
1. Autenrieth IB, Beer M, Bohn E, Kaufmann SHE, and
Heesemann J. Immune responses to Yersinia enterocolitica in
susceptible BALB/c and resistant C57BL/6 mice: an essential
role for gamma interferon. Infect Immun 62: 2590–2599, 1994.
2. Blanque R, Meakin C, Millet S, and Gardner CR. Dual
mechanisms of action of interferon-␥ in potentiating responses to
LPS in mice: IL-1, TNF-␣, and IL-6 production in serum and
hypothermia. Gen Pharmacol 32: 453–461, 1999.
3. Bohn E, Sing A, Zumbihl R, Bielfeldt C, Okamura H, Kurimoto M, Heesemann J, and Autenrieth IB. IL-18 (IFN-␥inducing factor) regulates early cytokine production in, and promotes resolution of, bacterial infection in mice. J Immunol 160:
299–307, 1998.
4. Conti B, Park LCH, Calingasan NY, Kim Y, Kim H, Bae Y,
Gibson GE, and Joh TH. Cultures of astrocytes and microglia
express interleukin 18. Mol Brain Res 67: 46–52, 1999.
5. Debets R, Timans JC, Churakowa T, Zurawski S, de Waal
Malefyt R, Moore KW, Abrams JS, O’Garra A, Bazan JF,
and Kastelein RA. IL-18 receptors, their role in ligand binding
and function: anti-IL-1RAcPL antibody, a potent antagonist of
IL-18. J Immunol 165: 4950–4956, 2000.
6. Dinarello CA. IL-18: a TH1 inducing proinflammatory cytokine
and a new member of the IL-1 family. Allergy Clin Immunol 103:
11–24, 1999.
7. Dinarello CA. Biologic basis for interleukin-1 in disease. Blood
87: 2095–2147, 1996.
8. Dinarello CA, Gatti S, and Bartfai T. Fever: the linkage of
prostaglandin E2, interleukin 1, and endotoxin signal transduction. Curr Biol 9: R147–R150, 1999.
9. Ek M, Arias C, Sawchenko P, and Ericsson-Dahlstrand A.
Distribution of the EP3 prostaglandin E2 receptor subtype in the
rat brain: relationship to sites of IL-1 induced cellular responsiveness. J Comp Neurol 428: 5–20, 2000.
10. Fantuzzi G and Dinarello CA. Interleukin 18 and IL-1␤: two
cytokine substrates for ICE (caspase 1). J Clin Immunol 19:
1–11, 1999.
11. Fantuzzi G, Puren AJ, Harding MW, Livingston DJ, and
Dinarello CA. Interleukin 18 regulation of interferon ␥ production and cell proliferation as shown in interleukin-1␤-converting
enzyme (caspase 1)-deficient mice. Blood 91: 2118–2125, 1998.
12. Garcia VE, Uyemura K, Sieling PA, Ochoa MT, Morita CT,
Okamura H, Kurimoto M, Rea TH, and Modlin RL. IL-18
promotes type 1 cytokine production from NK cells and T cells in
human intracellular infection. J Immunol 162: 6114–6121, 1999.
13. Gatti S and Bartfai T. Encyclopedia of Stress. San Diego, CA:
Academic, 2000, vol. 1, p. 116–123.
14. Ghayur T, Banerjee S, Hugunin M, Butler D, Herzog L,
Carter A, Quintal L, Sekut L, Talanian R, Paskind M,
Wong W, Kamen R, Tracey D, and Allen H. Caspase-1 processes IFN-␥-inducing factor and regulates LPS-induced IFN-␥
production. Nature 386: 619–623, 1997.
15. Goehler LE, Gaykema RPA, Nguyen KT, Lee JE, Tilders
FJH, Maier SF, and Watkins LR. IL-1␤ in immune cells of the
abdominal vagus nerve: a link between the immune and the
nervous system? J Neurosci 19: 2799–2806, 1999.
16. Grobmyer SR, Lin E, Lowry SF, Rivadeneira DE, Potter S,
Barie PS, and Nathan CF. Elevation of IL-18 in human sepsis.
J Clin Immunol 20: 211–215, 2000.
17. Guo F and Wu S. Antisense IRAK-1 oligonucleotide blocks
activation of NF-␬B and AP-1 induced by IL-18. Immunopharmacology 49: 241–246, 2000.
18. Hansen MK, O’Connor KA, Goehler LE, Watkins LR, and
Maier SF. The contribution of the vagus nerve in interleukin-1␤
fever is dependent on dose. Am J Physiol Regulatory Integrative
Comp Physiol 280: R929–R934, 2001.
19. Hansen MK, Taishi P, Chen Z, and Krueger JM. Vagotomy
blocks the induction of IL-1␤ mRNA in the brain of rats in
response to systemic IL-1␤. J Neurosci 18: 2247–2253, 1998.
20. Hochholzer P, Lipford GB, Wagner H, Pfeffer K, and Heeg
K. Role of interleukin-18 during lethal shock: decreased lipopolysaccharide sensitivity but normal superantigen reaction in IL18-deficient mice. Infect Immun 68: 3502–3508, 2000.
21. Hong T, Andersen NA, Nielsen K, Karlsen AE, Fantuzzi G,
Eizirik DL, Dinarello CA, and Mandrup-Poulsen T. Interleukin-18 mRNA, but not interleukin-18 receptor mRNA, is
constitutively expressed in islet ␤-cells and upregulated by interferon-␥. Eur Cytokine Netw 11: 193–205, 2000.
22. Kanaraj P, Ngo K, Wu Y, Angulo A, Ghazal P, Harris CA,
Siekierka JJ, Peterson PA, and Fung-Leung WP. Defective
IL-18 mediated natural killer and T helper cell type 1 responses
in IL-1 receptor associated kinase (IRAK) deficient mice. J Exp
Med 189: 1129–1138, 1999.
23. Kim SH, Eisenstein M, Reznikov L, Fantuzzi G, Novick D,
Rubistein M, and Dinarello CA. Structural requirements of
six naturally occurring isoforms of the IL-18 binding protein to
inhibit IL-18. Proc Natl Acad Sci USA 97: 1190–1195, 2000.
24. Kim SH, Reznikov LL, Stuyt RJL, Selzman CH, Fantuzzi
G, Hoshino T, Young HA, and Dinarello CA. Functional
reconstitution and regulation of IL-18 activity by the IL-18Rss
chain. J Immunol 166: 148–154, 2001.
25. Kohka H, Nishibori M, Iwagaki H, Nakaya N, Yoshino T,
Kobashi K, Saeki K, Tanaka N, and Akagi T. Histamine is a
potent inducer of IL-18 and IFN-␥ in human peripheral blood
mononuclear cells. J Immunol 164: 6640–6646, 2000.
26. Kojima H, Aizawa Y, Yanai Y, Nagaoka K, Takeuchi M,
Ohta T, Ikegami H, Ikeda M, and Kurimoto M. An essential
role for NF-kB in IL-18 induced IFN␥ expression in KG-1 cells.
J Immunol 162: 5063–5069, 1999.
27. Kojima H, Takeuchi M, Ohta T, Nishida Y, Arai N, Ikeda M,
Ikegami H, and Kurimoto M. Interleukin-18 activates the
IRAK-TRAF6 pathway in mouse EL-4 cells. Biochem Biophys
Res Commun 244: 183–186, 1998.
28. Kubota T, Fang J, Brown RA, and Krueger JM. Interleukin-18 promotes sleep in rabbits and rats. Am J Physiol Regulatory Integrative Comp Physiol 281: R828–R838, 2001.
29. McInnes IB, Gracie JA, Leung BP, Wei XQ, and Liew FY.
Interleukin 18: a pleiotropic participant in chronic inflammation. Immunol Today 21: 312–315, 2000.
30. Mee JB, Alam Y, and Groves RW. Human keratinocytes
constitutively produce but do not process interleukin-18. Br J
Dermatol 143: 330–336, 2000.
31. Micallef MJ, Yoshida K, Kawai S, Hanaya T, Kohno K, and
Arai S. In vivo antitumor effects of murine interferon-␥-inducing factor/interleukin-18 in mice bearing syngenic MethA sarcoma malignant ascites. Cancer Immunol Immunother 43: 361–
367, 1997.
32. Nakamura S, Otani T, Okura R, Ijiri Y, Motoda R, Kurimoto M, and Orita K. Expression and responsiveness of human
AJP-Regulatory Integrative Comp Physiol • VOL
282 • MARCH 2002 •
www.ajpregu.org
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.1 on June 17, 2017
In vivo studies on the neurological effects of cytokines of the IL-1 structural superfamily, like that by
Kubota et al. (28) or this study, could also contribute to
the effort of understanding the biology of the system. If
IL-18 is a modifier of IL-1␤ response, this effect could
be important, especially considering that IL-1␣/␤ are
the most potent among the pyrogenic, anorectic, and
somnogenic substances we know.
Fever, in particular, is a stereotyped and phylogenetically very ancient reaction to infection. The knowledge concerning other proinflammatory cytokines of
the IL-1 structural superfamily and their effect on
IL-1-induced fever is also of importance for our understanding of the molecular mechanisms inducing and
sustaining the pyrogenic response both in the periphery and at the hypothalamic level.
R709
IL-18 AND FEVER
33.
34.
35.
36.
37.
38. Reznikov LL, Kim SH, Westcott JY, Frishman J, Fantuzzi
G, Novick D, Rubinstein M, and Dinarello CA. IL-18 binding
protein increases spontaneous and IL-1-induced prostaglandin
production via inhibition of IFN-␥. Proc Natl Acad Sci USA 97:
2174–2179, 2000.
39. Sakao Y, Takeda K, Tsutui H, Kaisho T, Nomura F, Okamura H, Nakanishi K, and Akira S. IL-18 deficient mice are
resistant to endotoxin induced liver injury but highly susceptible
to endotoxin shock. Int Immunol 11: 471–480, 1999.
41. Tsuji-Takayama K, Aizawa Y, Okamoto I, Kojima H, Koide
K, Takeuchi M, Ikegami H, Ohta T, and Kurimoto M.
Interleukin-18 induces interferon-␥ production through NF-␬B
and NFAT activation in murine T helper type 1 cells. Cell
Immunol 196: 41–50, 1999.
42. Wheeler RD, Culhane AC, Hall MD, Pickering-Brown S,
Rothwell NJ, and Luheshi GN. Detection of the interleukin
18 family in rat brain by RT-PCR. Brain Res Mol Brain Res 77:
290–293, 2000.
43. Yamaguchi K, Motegi K, Kurimoto M, and Endo Y. Induction of the activity of the histamine-forming enzyme, histidine
decarboxylase, in mice by IL-18 and by IL-18 plus IL-12. Inflamm Res 49: 513–519, 2000.
AJP-Regulatory Integrative Comp Physiol • VOL
282 • MARCH 2002 •
www.ajpregu.org
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.1 on June 17, 2017
interleukin-18 receptor (IL-18R) on hematopoietic cell lines. Leukemia 14: 1052–1059, 2000.
Nakanishi K, Yoshimoto T, Tsutsui H, and Okamura H.
Interleukin-18 is a unique cytokine that stimulates both Th1 and
Th2 responses depending on its cytokine milieu. Cytokine
Growth Factor Rev 12: 53–72, 2001.
Netea MG, Kullberg BJ, Verschueren I, and Van Der Meer
JW. Interleukin 18 induces production of proinflammatory cytokines in mice: no intermediate role for the cytokines of the tumor
necrosis factor family and interleukin-1␤. Eur J Immunol 30:
3057–3060, 2000.
Prinz M and Hanisch UK. Murine microglial cells produce and
respond to interleukin 18. J Neurochem 72: 2215–2218, 1999.
Puren AJ, Fantuzzi G, and Dinarello CA. Gene expression,
synthesis, and secretion of interleukin 18 and interleukin 1␤ are
differentially regulated in human blood mononuclear cells and
mouse spleen cells. Proc Natl Acad Sci USA 96: 2256–2261,
1999.
Puren AJ, Fantuzzi G, Gu Y, Su MSS, and Dinarello CA.
Interleukin 18 induces IL-8 and IL-1␤ via TNF␣ production from
non-CD14⫹ human blood mononuclear cells. J Clin Invest 101:
711–721, 1998.