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converting enzyme resist
anorexia induced by central lipopolysaccharide
Laboratories of
1 Integrative Biology and
2 Immunophysiology, Interleukin-1
food intake; behavior; cytokine
INTERLEUKIN-1 Previous studies in mice involving intraperitoneal or
intracerebroventricular administration of recombinant IL-1 Because ICE Reagents
ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(IL-1) is expressed in the
mouse brain after intracerebroventricular injection of
lipopolysaccharide (LPS) and is thought to be responsible for many of
the behavioral and neuroendocrine changes that occur during
inflammation. In this study we show that LPS in the brain also induces
expression of interleukin-1 converting enzyme (ICE) and that ICE is
important for the characteristic anorectic response of mice to
intracerebroventricular LPS. Specifically, mice that were deficient in
ICE (ICE
/)
resisted the anorexia caused by intracerebroventricular injection of
LPS but were sensitive to the anorectic properties of recombinant IL-1. The typical anorectic response seen in wild-type (WT) mice after LPS was restored in
ICE/ mice by
intracerebroventricular administration of the ICE analog cathepsin G. Conversely, anorexia induced by intracerebroventricular injection of
LPS in WT mice was blocked by prior intracerebroventricular injection
of the ICE antagonist YVAD.CMK. Furthermore, in situ hybridization
immunohistochemistry revealed intense expression of ICE mRNA in the
hippocampus and dorsomedial hypothalamus of WT mice after
intracerebroventricular injection of LPS. Thus ICE mRNA is expressed in
brain after intracerebroventricular injection of LPS and is important
for induction of anorexia, presumably because it generates mature
IL-1. These results suggest that preventing generation of mature
IL-1 can inhibit anorexia induced by LPS in the brain and,
therefore, reveal ICE as a potential target for regulating food intake
during brain inflammation.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(IL-1) is
synthesized as a 31-kDa protein that has little affinity for either the
type I or type II IL-1 receptor and therefore has only marginal
biological activity (8). It must be cleaved by
interleukin-1 converting enzyme (ICE) to produce the
mature biologically active 17-kDa form of IL-1 (1,
38). Cells that do not express ICE are unable to release biologically active IL-1 (6). Accordingly, in the presence of competitive substrate ICE inhibitors, the cleavage and release of IL-1 is reduced (22, 24, 25, 38). Moreover, cultured macrophages from
ICE-deficient mice (ICE/) do not
release mature (i.e., biologically active) IL-1 after stimulation
with lipopolysaccharide (LPS) in vitro (22, 24), and serum
concentrations of IL-1 as well as IL-1
, tumor necrosis factor-
(TNF-), interferon-
(IFN-), and IL-18 are decreased compared
with wild-type (WT) mice (13, 14, 22, 24). The fact that the expression
of multiple proinflammatory cytokines is altered in
ICE/ mice and
ICE/ mice are
notably resistant to the lethal effects of LPS make ICE a good
candidate for inhibiting inflammatory responses.
indicate that this cytokine induces many components of the central acute phase
response, including anorexia (10, 31). Both intraperitoneal and
intracerebroventricular injection of LPS reduce motivation for food and
induce IL-1 mRNA synthesis in the brain (34, 36). Moreover, rats
bearing prostate adenocarcinomas were anorectic and had increased
IL-1 mRNA in brain (33). The IL-1 produced and released in the
brain (as opposed to systemically) may be directly responsible for
anorexia, because IL-1 receptor antagonist infused into the brain but
not intraperitoneally blocked anorexia caused by experimentally induced
colitis (27). Therefore, a better understanding of the cellular and
molecular mechanisms involved in the induction, synthesis, and release
of IL-1 in the brain is needed to treat or prevent anorexia induced
by inflammation.
/ mice
are unable to produce biologically active IL-1 (22, 24) they provide
a unique model to explore the importance of generating mature IL-1
in the brain in LPS-induced anorexia. We recently reported that
ICE/ mice were
anorectic after systemic injection of LPS but that they were not
anorectic when LPS was administered intracerebroventricularly (5). This
study suggested that generation of IL-1 after
intracerebroventricular administration of LPS is a prerequisite for
induction of anorexia. In the present study, we further explored this
hypothesis by comparing food intake in WT and
ICE/ mice after
intracerebroventricular injection of increasing doses of LPS. In
addition, we determined if anorexia induced by
intracerebroventricularly administered LPS in WT mice could be blocked
by inhibiting ICE within the central nervous system (CNS) and if the
normal anorectic response to intracerebroventricularly administered LPS
could be restored in
ICE/ mice by
intracerebroventricular administration of an ICE analog. Because murine
microglia express ICE mRNA and protein in response to LPS in vitro
(39), we also determined by in situ hybridization immunohistochemistry
the location and intensity of ICE mRNA expression in brain after
intracerebroventricular injection of LPS in WT mice. The results show
that ICE mRNA is expressed in brain after intracerebroventricular
injection of LPS and that ICE is important for induction of anorexia.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
was
purchased from Pharmingen (San Diego, CA). It reportedly contained
<0.1 ng of residual endotoxin per microgram of IL-1. Murine
IL-1 was dissolved in water and then in PBS with 3% BSA (detoxified
tissue culture grade; Stem Cell Technologies, Vancouver, BC, Canada)
and stored frozen at 
80°C in 5-µl aliquots containing 50 ng of IL-1. Before use, an aliquot was brought to room temperature
and diluted to the desired concentration with PBS.
Animals
Male 12- to 16-wk-old ICE gene knockout (ICE/) mice and
their age-matched WT inbred controls were used in all experiments (24). All mice were obtained from an in-house colony that was established from ICE/ and WT
mice provided by Dr. Tara Shashadri at BASF Bioresearch (Worcester, MA) (24). Mice were kept in groups of three in
polypropylene cages until ~10 wk of age, when they were housed
individually in modified metabolic cages. They were maintained at
25°C under a reverse 12:12-h light-dark cycle (lights on at 1900)
with ad libitum access to water and rodent chow. All procedures were
approved by the University of Illinois Committee for the Care and Use
of Laboratory Animals.
Intracerebroventricular Cannulation and Injection
Each mouse was anesthetized (ketamine 61 mg/kg ip and xylazine 9 mg/kg ip) for implantation of a cannula in the left lateral cerebral ventricle. The head of each mouse was oriented in a stereotaxic instrument (David Kopf Instrument, Tujunga, CA) so that the plane formed by the frontal and parietal bones was parallel to the instrument table top. A 26-gauge stainless-steel guide cannula (Plastics One, Roanoke, VA) was placed intracerebroventricularly using predetermined coordinates (anteroposterior0.6 mm; lateral, 1.6 mm to the bregma; horizontal 2.0 mm to the dura
mater) and secured with two stainless steel screws and
cranioplastic cement. When not in use, a dummy cannula was inserted
into the guide cannula to prevent occlusion and infection. At least 7 days were allowed for recovery before treatments were initiated.
All intracerebroventricular treatments were delivered through the implanted cannula in a 2 µl volume over a 30-s period using a 33-gauge injection cannula and a syringe pump (World Precision Instruments, Sarasota, FL). After an experiment, trypan blue was injected via the guide cannula, and the brains were dissected to verify cannula position within the lateral ventricle. Data from mice determined to have incorrect placement of the intracerebroventricular cannula were excluded.
In Situ Hybridization
Generation of RNA probe. Total RNA was isolated from cultured N13 microglial cells that had been stimulated with LPS (39). RNA was reverse transcribed, and a 400-bp region of ICE was amplified by PCR using oligonucleotide primers and techniques previously described (39). The PCR-generated cDNA product was ligated into pCR II vectors according to the manufacturer's instructions using a TA cloning kit (Invitrogen, Carlsbad, CA). The ligation mix was then used to transform competent cells, and transformants were selected on Luria-Bertani agar plates containing X-Gal and ampicillin. DNA purified from positive clone (prepared using the Plasmid Midi kit, QIAGEN) was subjected to dideoxy sequencing to authenticate the murine ICE cDNA. Subsequently, plasmid DNA was generated on a large scale and subjected to restriction enzyme digestion to produce linearized DNA, which was purified and subjected to in vitro transcription using a MAXIscript kit (Ambion, Austin, TX). Sense and antisense RNA probes were labeled by incorporation of digoxigenin (DIG)-UTP (Boehringer Mannheim) during in vitro transcription (DIG-UTP:UTP = 3:7).Preparation of brain tissue. Mice were
deeply anesthetized with halothane, perfused transcardially through the
left ventricle with diethyl pyrocarbonate (DEPC)-treated
PBS (containing 10 U/ml heparin) for 15 min and with 4%
paraformaldehyde-DEPC for another 15 min. Brains were carefully removed
and postfixed by immersion in 4% paraformaldehyde-DEPC for 2 h at
4°C before they were transferred to 30% sucrose-DEPC for 2 days at
4°C. Brains were embedded in OCT compound (Sakura, Japan) and
stored at 80°C until sectioned. Coronal sections (20 µm)
were taken at the level of the posterior hypothalamus using a
Reichert-Jung Cryocut 1800 (McHenry, Illinois). Sections were mounted
onto aminosilane-coated slides (Newcomer Supply, Middleton, WI) and
processed for in situ hybridization or dried and stored at
80°C.
Hybridization. To improve probe and antibody penetration, 0.1% Brij (Sigma Chemical, St. Louis, MO) was included in all solutions. Slides were washed in PBS (5×) and incubated with proteinase K (5 µg/ml) for 30 min at 37°C. After washing with PBS (5×), adjacent slides were hybridized with prehybridization buffer for 1 h at 37°C and hybridized at 50°C overnight with 1) antisense RNA probe labeled with DIG-UTP, 2) sense RNA probe labeled with DIG-UTP, or 3) antisense RNA probe without DIG-UTP labeling. After hybridization, slides were washed with 2× SSC solution (5×) (1× SSC is 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0) and treated with RNase A (20 µg/ml in 2× SSC) for 30 min at 37°C. Then slides were washed sequentially with 1× SSC (5×) and 0.1× SSC (6×) at room temperature.
Immunological detection. Immunological detection was carried out using a DIG nucleic acid detection kit (Boehringer Mannheim). Slides were washed with PBS (5×) and blocked in 1% Boehringer Block solution for 60 min. Slides were incubated with alkaline phosphatase-labeled anti-DIG-AP antibody (1:250) overnight at 4°C. Unbound antibody was removed by several washes, and slides were incubated with freshly prepared color substrate (NBT/BCIP) for up to 16 h, when the reaction was stopped in stopping buffer (10 mM Tris · HCl, 1 mM EDTA, pH 8.0). Labeled sections were mounted in glycerol gel and analyzed by light microscopy.
Protocol
Central injection of LPS and IL-1 in
WT and ICE/
mice. WT and
ICE/ mice were
injected intracerebroventricularly with PBS or PBS containing 1, 10, or
100 ng LPS. Mice were weighed, and injections were given at 0830, which
was 30 min before the onset of darkness. After injection, food was
available ad libitum. Feed cups were weighed before
intracerebroventricular injection (0 h) and at 2, 4, 8, and 12 h
postinjection so that food intake could be calculated. At least two
separate but otherwise identical trials were conducted, and each
treatment was replicated a minimum of six times. In a separate but
similar study, WT and
ICE/ mice were
injected intracerebroventricularly with vehicle or 2 ng recombinant
murine IL-1. Food intake was monitored as previously described, and
each treatment was replicated a minimum of six times over two trials.
Effects of cathepsin G and YVAD.CMK on in vitro
secretion of IL-1 by macrophages from WT and
ICE/
mice. Elicited peritoneal macrophages were obtained
from WT and ICE/
mice 3 days after intraperitoneal injection of 1 ml 5% thioglycollate broth. Cells were collected aseptically by lavage of the peritoneal cavity with RPMI 1640 containing 10 U/ml heparin. Cells were washed, resuspended in RPMI 1640 medium (supplemented with 10% FBS, 2 g/l
sodium bicarbonate, 100 U/ml penicillin G, and 100 g/ml streptomycin), and plated at 106 cells/well in
24-well, flat-bottom culture plates for 1 h at 37°C with 95%
humidity and 7% CO2. After
removing nonadherent cells by repeated washing, triplicate wells of
macrophages from ICE/ mice were
incubated 6 h with medium alone, medium containing 100 ng/ml LPS, and
medium containing 100 ng/ml LPS plus 5 U/ml cathepsin G. Macrophages
from WT mice were incubated with vehicle or 5 µM YVAD.CMK for 1 h.
Vehicle or 100 ng/ml LPS was then added, and cells were incubated for 6 h. Supernatants were collected and stored at 80°C until
assaying for IL-1 using an ELISA kit specific for murine IL-1
(Endogen, Cambridge, MA). Each treatment was replicated a minimum of
nine times. No treatment was found to affect cell viability as
determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.
Effects of YVAD.CMK and cathepsin G on food intake of
WT and ICE/ mice
injected intracerebroventricularly with
LPS. WT mice were prepared with an
intracerebroventricular cannula, and the ICE inhibitor YVAD.CMK was
administered before LPS. YVAD.CMK was administered in two sequential
intracerebroventricular injections as described by Hara et al. (15).
Briefly, mice were injected with vehicle or 100 ng YVAD.CMK. Thirty
minutes later, mice received a second injection of vehicle, vehicle
containing either LPS (1 ng), YVAD.CMK (100 ng), or both. Thus the four
treatments included vehicle, LPS, YVAD.CMK, and YVAD.CMK+LPS. The
second injection was administered at 0830, and food intake was measured
4 h postinjection. Each treatment was replicated a minimum of six times
over two trials.
In a separate but similar experiment,
ICE/ mice were
injected intracerebroventricularly with vehicle or 2 U cathepsin G, 1 ng LPS, or both. Thus the four treatments included vehicle, LPS, cathepsin G, and cathepsin G+LPS. Treatments were administered 30 min
before the onset of darkness (0830) and food intake was measured 4 h
postinjection. Each treatment was replicated a minimum of six times
over two trials.
ICE mRNA expression in brain after
intracerebroventricular injection of LPS. Because there
are no full-length ICE transcripts in various organs, including brain
in ICE/ mice (24),
only WT mice were used in this experiment. WT mice prepared with an
intracerebroventricular cannula were injected with PBS alone or PBS
containing 1 or 100 ng LPS (n = 3).
Two or four hours after injection, mice were deeply anesthetized by halothane, perfused transcardially with DEPC-treated PBS (containing 10 U/ml heparin) for 15 min, and fixed with 4% paraformaldehyde-DEPC for
another 15 min. Brains were carefully removed, and coronal sections
were prepared for in situ hybridization immunohistochemistry as
previously described. Brain sections from the different groups of mice
were matched for rostrocaudal levels as closely as possible using the
mouse brain atlas of Franklin and Paxinos (10a).
Statistical Analysis
All data analysis was conducted using general linear model procedures (35). Metabolic body size (MBS) was calculated for each mouse (body weight in kg raised to the three-fourths power; Ref. 7) and food intake was expressed as grams consumed per unit MBS. Data were subjected to two-way (LPS × time, genotype × IL-1, LPS × YVAD.CMK, LPS × cathepsin G) or three-way (genotype × LPS × time) ANOVA to determine the significance of main factors and
the main factor interaction. When ANOVA revealed a significant effect
of dose or a dose × main factor interaction, differences between
treatment means were tested using least squares difference tests. Data
are reported as the treatment means ± SE.
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RESULTS |
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ICE/ Mice Are Resistant to Anorexia
Caused by Central LPS
/) was
compared after intracerebroventricular injection of LPS. Three-way
ANOVA of food intake revealed a significant effect of genotype
(P < 0.0001), treatment
(P < 0.0001), time
(P < 0.0001), a genotype × treatment interaction (P < 0.0001),
a genotype × time interaction (P < 0.0001), a time × treatment interaction (P < 0.0001), and a genotype × treatment × time interaction (P < 0.001). The effects of intracerebroventricular LPS on food intake for both WT and ICE/
mice are shown in Fig. 1. As anticipated,
LPS decreased food intake by WT mice in a time- and dose-dependent
fashion. Compared with PBS controls, at 4 h postinjection food intake
by WT mice receiving 1, 10, and 100 ng LPS was reduced by 85, 92, and
98%, respectively. In contrast,
ICE/ mice were
highly resistant to the anorectic properties of LPS. At 4 h
postinjection, even the 100 ng dose of LPS did not depress food intake
by mice deficient in ICE. Although food intake of ICE/ mice receiving
10 or 100 ng LPS was depressed from 4-8 h postinjection, those
receiving 1 ng LPS continued to consume an amount of food similar to
that of the PBS-injected controls. In a separate but similar study, WT
and ICE/ mice were
injected intracerebroventricularly with recombinant murine IL-1, and
food intake was measured 4 h postinjection. Two-way ANOVA (treatment × genotype) of 4-h food intake revealed a significant effect of
treatment (P < 0.001), but neither
genotype nor the genotype × treatment interaction was significant
(P > 0.05). As evident in Fig.
2, IL-1 injected
intracerebroventricularly depressed food intake similarly in both WT
and ICE/ mice.
Collectively, these results suggest that ICE has a pivotal role in the
anorexia caused by LPS in the brain, probably because it processes
pro-IL-1 to its mature biologically active form.
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An ICE Analog Restores the Anorectic Properties of LPS in
ICE/ Mice
three
amino acids from the ICE site and generate an IL-1-like molecule
that has biologic activity similar to authentic IL-1 (16).
Therefore, it stands to reason that the typical anorectic response to
LPS in the brain could be restored in
ICE/ mice by
administering cathepsin G. However, before testing this idea in vivo,
we sought to determine if cathepsin G could process pro-IL-1 in
peritoneal macrophages isolated from
ICE/ mice.
Macrophages from
ICE/ mice were
incubated with medium alone or medium containing LPS, cathepsin G, or
both. Two-way ANOVA of supernatant IL-1 concentration revealed a
significant LPS cathepsin G interaction
(P < 0.05). Neither LPS nor
cathepsin G alone stimulated IL-1 secretion by macrophages deficient
in ICE (IL-1 concentration of supernatants from macrophages treated
with medium, LPS, or cathepsin G was 4.7 ± 2.34, 5.3 ± 1.90, and 6.5 ± 1.66 pg/ml, respectively;
P > 0.05). However, culturing
peritoneal macrophages from
ICE/ mice with both
LPS and cathepsin G resulted in a marked increase in supernatant
IL-1 concentration (23.9 ± 6.79 pg/ml). Because the ELISA was
specific for mature IL-1 (18), these data indicate that cathepsin G
can process pro-IL-1 at least in cells that are derived from
mononuclear myeloid progenitors and that lack ICE.
To determine if cathepsin G could restore the anorectic properties of
central LPS in ICE/
mice, cathepsin G and LPS were injected intracerebroventricularly in
ICE/ mice, and food
intake was assessed. The 1 ng dose of LPS was used because in the first
study it induced a robust anorectic response in WT mice but not in
ICE/ mice (Fig. 1).
Two-way ANOVA revealed a significant LPS × cathepsin G
interaction (P < 0.05). Given alone,
neither LPS nor cathepsin G reduced food intake compared with PBS
controls (P > 0.05; Fig. 3). However, when cathepsin G and LPS were
coadministered intracerebroventricularly, food intake was markedly
depressed (Fig. 3).
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An ICE Inhibitor Blocks the Anorexia Caused by Central LPS in WT Mice
To determine if inhibition of ICE attenuates LPS-induced anorexia, WT mice were injected intracerebroventricularly with YVAD.CMK or vehicle and 30 min later with the same inhibitor plus LPS or PBS. YVAD.CMK inhibits IL-1 release by binding the active site of ICE (29). The fact that YVAD.CMK could inhibit ICE activity was
verified in vitro by treating peritoneal macrophages cultured from WT
mice with medium alone or medium containing LPS, YVAD.CMK, or both.
Two-way ANOVA of supernatant IL-1 concentration revealed a
significant LPS × YVAD.CMK interaction
(P < 0.05). LPS induced a marked
increase in IL-1 secretion by peritoneal macrophages from WT mice.
However, the LPS-induced secretion of IL-1 was abrogated by YVAD.CMK
(IL-1 concentration of supernatants from macrophages treated with
medium, YVAD.CMK, LPS, or YVAD.CMK+LPS was 20.3 ± 3.18, 17.5 ± 2.43, 109.0 ± 4.39, 63.4 ± 7.10 pg/ml, respectively). Consistent with the idea that ICE is important for the
induction of anorexia by central LPS, YVAD.CMK administered intracerebroventricularly completely blocked the hypophagia caused by
intracerebroventricular injection of LPS (Fig.
4). Two-way ANOVA revealed a significant
LPS × YVAD.CMK interaction (P < 0.05).
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Intracerebroventricular LPS Induces ICE mRNA Expression in the Brain
We previously reported that LPS induced ICE mRNA and protein expression in microglial cells in vitro (39). The previous studies clearly showed that ICE plays a pivotal role in the depression of food intake induced by intracerebroventricular LPS. However, where in the brain ICE is expressed is not known. In this study WT mice were injected intracerebroventricularly with PBS or LPS (1 or 100 ng), and the location and intensity of ICE mRNA expression on coronal sections obtained from brains collected 2 or 4 h postinjection were visualized by in situ hybridization immunohistochemistry. Brain sections from the different groups of mice were matched for rostrocaudal levels as closely as possible and were taken at a level that included the posterior hypothalamus and the hippocampus. With the DIG-labeled antisense RNA probe and immunohistochemical detection, a marked increase in ICE mRNA was evident after LPS in every brain area examined. However, two areas densely populated with cells expressing ICE mRNA were evident, the hippocampus and the dorsomedial hypothalamus. Figure 5 shows the effects of LPS on the expression of ICE mRNA in the hippocampus of WT mice. Compared with PBS control, LPS increased ICE mRNA at 2 and 4 h. The intensity of ICE mRNA expression was greatest at 4 h and occurred in a dose-dependent fashion. This is apparent in Fig. 6, which shows a higher magnification of the CA3 field.
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Figure 7 demonstrates the intense
expression of ICE mRNA in the posterior hypothalamus 4 h after
intracerebroventricular injection of PBS or LPS (1 or 100 ng).
Increased ICE mRNA is evident in the periventricular nucleus, as might
be expected because LPS was given intracerebroventricularly. Also
evident is a dense population of cells that are positive for ICE mRNA
and that map to the dorsomedial hypothalamus. As in the hippocampus,
the intensity of ICE mRNA expression is dose dependent.
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In addition to the DIG-labeled anti-sense RNA probe used to detect ICE mRNA, a DIG-labeled sense RNA probe and an unlabeled antisense RNA probe were included as controls. There was little to no signal detected when sections were hybridized with the unlabeled antisense RNA probe (data not shown). After injection of PBS or LPS there also was little to no signal detected when sections were hybridized with the DIG-labeled sense RNA probe (not shown).
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DISCUSSION |
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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IL-1 is synthesized as an inactive 31-kDa precursor molecule with
little to no biological activity. ICE cleaves the precursor protein
between Asp116 and Ala117 to generate and stimulate release of the
biologically active 17-kDa form of IL-1 (38). Neurons, astrocytes,
and microglia express ICE and IL-1 (3, 17, 23, 37, 39, 40), and the
IL-1 that is produced in the brain is considered important in
regulating food intake in people and animals with acute and chronic
diseases (32, 33). Neurons and glia, however, also produce a number of
other cytokines, such as TNF- and IL-6, which can induce anorexia.
Therefore, to better describe the role of brain IL-1 in food intake
regulation during sickness, in this study both WT and
ICE/ mice were
injected intracerebroventricularly with LPS. Consistent with previous
studies, WT mice that were capable of generating mature IL-1 showed
marked anorexia even when the lowest dose of LPS was administered.
However, ICE/ mice
were not hypophagic after intracerebroventricular injection of the same
low dose of LPS. Because pro-IL-1 is the primary substrate of ICE,
this suggests that IL-1 is a critical cytokine for anorexia induced
by intracerebroventricular LPS. It is possible, however, that cytokines
other than IL-1 influenced the anorectic response, because
ICE/ mice injected
intraperitoneally with LPS had reduced circulating levels of not only
IL-1, but also IL-1, IL-18, and TNF- (13, 14, 22, 24).
Furthermore, the reduction in food intake by IL-1-deficient mice
challenged intraperitoneally with LPS was similar to that by WT
controls (21), indicating that secretion of IL-1 is not always a
prerequisite for LPS-induced anorexia. Indeed, in the present study
ICE/ mice were
hypophagic 4-8 h after injection of 10 or 100 ng LPS, suggesting
that other cytokines became involved. Nonetheless, because
ICE/ mice were
completely resistant to the anorectic properties of a low dose of LPS
and showed an attenuated anorectic response to higher doses suggests
IL-1 is a critical cytokine for food intake regulation, at least
when inflammatory stimuli are localized in the CNS.
Although ICE generates authentic IL-1 by cleaving pro-IL-1
between Asp116 and Ala117, other enzymes generate bioactive
IL-1-like molecules from the same precursor (4, 16, 20, 26, 28). For
instance, an IL-1-like molecule generated by cleavage of pro-IL-1
between Tyr113 and Val114 was found in the bronchoalveolar lavage fluid
from patients with active sarcoidosis (16). Cathepsin G was
consistently detected in these patients and was proposed to be the
proteolytic enzyme responsible for generation of the IL-1-like
molecule (16). Consistent with these findings, in the present study
when macrophages isolated from
ICE/ mice were
treated with LPS alone there was little to no increase in supernatant
IL-1 concentration. However, when peritoneal macrophages were
treated with both LPS and cathepsin G, a marked increase in supernatant
IL-1 concentration was evident.
We took advantage of this system by coadministering cathepsin G and LPS
intracerebroventricularly in
ICE/ mice to further
evaluate the importance of IL-1. On the basis of results from the
first study, the 1 ng dose of LPS was used and food intake was
determined 4 h after injection. Consistent with the hypothesis that
mature IL-1 is needed for this dose of LPS in the brain to induce
anorexia, ICE/ mice
resisted anorexia when LPS was given alone. However, when LPS and
cathepsin G were coadministered, marked anorexia ensued, presumably
because IL-1-like molecules were produced. Although beyond the scope
of this study, because cathepsin G increases cell membrane permeability
(30), it may have processed pro-IL-1 intra- or extracellularly. For
instance, putative membrane channels that allow passive movement of
pro-IL-1 and mature IL-1 have been proposed (8). When ICE
activity is blocked with a reversible competitive substrate inhibitor,
greater amounts of pro-IL-1 are found in the cell supernatants (38).
On stimulation with LPS, pro-IL-1 accumulates in cells from
ICE/ mice (22, 24).
Therefore, it is possible that intracerebroventricular injection of LPS
increased synthesis of pro-IL-1 that accumulated intracellularly and
that cathepsin G increased cell membrane permeability and caused the
extracellular release of pro-IL-1, which was subsequently cleaved.
The fact that other enzymes besides ICE can cleave pro-IL-1 in vivo
has recently been confirmed, because plasma IL-1 was similar in WT
and ICE / mice after
sterile tissue damage caused by turpentine (9).
Because resistance to LPS-induced anorexia in
ICE/ mice was
overcome by administration of an ICE analog, it followed that
inhibition of ICE in WT mice should prevent anorexia induced by central
LPS. YVAD.CMK is a competitive inhibitor of ICE and has been used in vivo to decrease active IL-1 and brain damage after ischemia (15). In the present study, the dose and injection regimen, which
involved two intracerebroventricular injections of YVAD.CMK spaced 30 min apart, was patterned after Hara et al. (15). The results showed
that inhibiting ICE with YVAD.CMK blocked the anorexia induced by LPS
in the brain and, therefore, are consistent with the results of the
previous studies. Because YVAD.CMK is specific for ICE and therefore
should not have interfered with the production of other cytokines known
to decrease appetite, these data further suggest a dominant role of
IL-1 in regulating food intake after central injection of LPS.
In a previous study, we found that LPS induced ICE mRNA and protein in
murine microglia in vitro (39), which was consistent with a report
showing that microglia were immunoreactive for ICE after global
ischemia (3). In the present study, to determine if LPS also
induced ICE mRNA by CNS cells in vivo, in situ hybridization immunohistochemistry was performed. The results showed that LPS induced
a time- and dose-dependent increase in ICE mRNA expression in the brain
areas examined. However, the dorsomedial hypothalamus and hippocampus
comprised a dense population of cells expressing ICE mRNA.
Interestingly, these brain areas regulate a number of behavioral and
physiological processes that are affected by IL-1, including
feeding, learning, and memory. For example, the dorsomedial hypothalamic nucleus (DMN) is an important nodal point in
neuroendocrine and autonomic homeostasis in that it receives
projections from the ventromedial hypothalamic nucleus (VMN) and
projects to the paraventricular nucleus of the hypothalamus (2). The
DMN is composed of cells and fibers containing neuropeptide Y (NPY), and the nutritional status (starvation-refeeding) is reflected in NPY
levels of both the VMN and DMN (2). Recently, IL-1 has been shown to
inhibit the synthesis of NPY (12).
The fact that there appeared to be discretion, at least in the density
of cells staining for ICE mRNA, suggests that this approach may provide
important insight into the specific role of IL-1 in the brain.
However, more work is needed to neuroanatomically map ICE expression in
brain. Determining the cell type expressing ICE after injection of LPS
was beyond the scope of this study. However, we predicted that
microglia would be the primary cell expressing ICE because
1) microglia are the major source of
IL-1 in the brain, 2) microglia
expressed ICE mRNA and protein in response to LPS in vitro (39), and
3) Bhat et al. (3) found in gerbils that both neurons and microglia had low immunoreactivity to ICE protein
in the hippocampus, but only microglia specifically increased their
immunoreactivity to ICE in response to global forebrain ischemia. Nonetheless, that in situ hybridization
immunohistochemistry revealed a pattern of ICE mRNA expression
consistent with a neuronal source cannot be ignored. Interestingly,
recent studies showed that intracerebroventricular injection of LPS
increased IL-1 mRNA expression in the hypothalamus and hippocampus
(11), which is consistent with the present finding that mRNA encoding
for the enzyme needed to produce biologically active IL-1 is also expressed in these areas. However, we cannot exclude the possibility that ICE may have other functions besides cleaving pro-IL-1 that are
yet to be described.
Perspectives
In this study we show that LPS in the brain induces expression of ICE and that inhibition of ICE is sufficient to make mice resistant to anorexia. Because pro-IL-1 is the substrate of ICE, these results
suggest that preventing generation of mature IL-1 in the brain can
inhibit anorexia. This might be important because anorexia as well as
IL-1 expression in brain has been documented in several infectious,
autoimmune, and neoplastic diseases. Therefore, ICE in brain is a
potential target for regulating food intake at times of disease.
However, the utility of inhibiting ICE is not yet clear because
IL-1-deficient mice and
ICE/ mice are fully
responsive to the anorectic properties of inflammatory stimuli in the periphery.
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ACKNOWLEDGEMENTS |
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This research was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-51576 (to R. W. Johnson).
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FOOTNOTES |
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Address for reprint request and other correspondence: R. W. Johnson, 390 Animal Sciences Laboratory, 1207 West Gregory Dr., Urbana, IL 61801.
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 20 April 1999; accepted in final form 8 July 1999.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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