Yonago Acta medica 2003;46:49–55

Angiotensin-Converting-Enzyme Inhibitor, Lisinopril, Reduces Lipopolysaccharide-Induced Expression of SplenicInterleukin-6 mRNA in Dehydrated Rats
Tatsuo Watanabe, Michio Miyoshi, Katsumi Nagata and Toshiaki Imoto
Division of Integrative Physiology, Department of Functional, Morphological and Regulatory
Science, Tottori University Faculty of Medicine, Yonago 683-8503 Japan
Angiotensin II (ANG II) has been shown to have proinflammatory properties. To investigate
whether ANG II is involved in the lipopolysaccharide (LPS)-induced production of apyrogenic/proinflammatory cytokine, interleukin-6 (IL-6), we examined the effects of anangiotensin-converting-enzyme (ACE) inhibitor, lisinopril, on LPS-induced fever and on
the expression of IL-6 mRNA in the spleen of dehydrated rats (in which the secretion of
ANG II increases). The results showed that the ACE inhibitor significantly inhibited
LPS-induced fever as well as the splenic expression of IL-6 mRNA in dehydrated rats. It
is suggested that endogenous ANG II may be involved in the production of IL-6 that occurs
in response to LPS, and thereby contribute to the LPS-induced febrile response in dehydrated rats.
Key words: angiotensin-converting-enzyme inhibitor; cytokine; interleukin-6; lipopolysaccharide;
spleenAngiotensin II (ANG II), a bioactive peptide wellknown to play an important role in blood-pressure
and body-fluid regulation, seems to participate ininflammatory responses, too. For example, ANG II
induces an inflammatory response—involving increases in the expressions of such proinflammatory
enzymes as phospholipase (Schlondorff et al., 1987)
and NADPH oxidase (Griendling et al., 1994)—and
ANG-II-type-1 (AT1) receptors are involved in certain types of cardiovascular inflammation (Usui etal., 2000). Furthermore, angiotensin-convertingenzyme (ACE) inhibitors have an anti-inflammatory effect (Delfraissy et al., 1984; Martin et al.,
1984; Rezkalla et al., 1990). Recently, we reported
results suggesting that ANG II is involved in thedevelopment of the fever (another example of an inflammatory response) induced by lipopolysaccha-ride (LPS) in rats (Watanabe et al., 2000). In fact,
the LPS-induced fever seen by us in rats was significantly attenuated by an ACE inhibitor. Since, as
the first step in fever induction, the pyrogenic/proinflammatory cytokine interleukin-1 (IL-1) is released from macrophages following their stimulation with LPS (Dinarello, 1984; Kluger, 1991), we
next examined the effect of the same ACE inhibitoron the fever induced by IL-1. However, this fever
underwent no alterations with the ACE inhibitor(Watanabe et al., 2000), so we concluded that ANG
II contributes to the LPS-induced production of IL1 in rats.
It has repeatedly been suggested that the LPSinduced production of IL-1 in the tissues results inthe local induction of IL-6 and that this then enters
the general circulation to cause fever (Kluger, 1991;

Abbreviations: ACE, angiotensin-converting-enzyme; ANG II, angiotensin II; ANOVA, analysis of variance; AT1,
ANG-II-type-1; IL-1, interleukin-1; IL-6, interleukin-6; i.p., intraperitoneal(ly); i.v., intravenous(ly); LPS,
lipopolysaccharide;49

T. Watanabe et al.

Luheshi et al., 1997; Luheshi, 1998). For thatreason, IL-6 is now thought to be a candidate for acirculating pyrogenic cytokine (Kluger, 1991).
Moreover, there is an undetectable amount of IL-1β
in the blood of animals treated with LPS (Kluger,
1991). Taking the above evidence together, it seems
likely (i) that ANG II participates in the productionof tissue IL-1 in response to LPS and (ii) that the
local induction of IL-6 by this IL-1 might be, atleast in part, involved in the development of the
LPS-induced fever after the entering general circulation and acting on the brain as a circulating pyrogenic cytokine.
The present study was carried out to investigate whether the LPS-induced production of IL-6
does indeed involve an action by ANG II. To thisend, the effects of an ACE inhibitor on LPSinduced fever and on the expression of IL-6 mRNAin the spleen, a representative organ of the reticuloendothelial system, were examined in dehydrated
rats. Dehydrated rats (in which the secretion of
ANG II increases) were chosen, because, in our previous study (Watanabe et al., 2000), although the
LPS-induced fever was significantly attenuated by
an ACE inhibitor in both dehydrated and euhydrated rats, the effect was greater in the former. The
present results showed that ACE inhibition significantly inhibited both LPS-induced fever and thesplenic expression of IL-6 mRNA in dehydrated
rats, suggesting that ANG II is involved in the LPSinduced production of IL-6 in such rats.

cols were reviewed by the Committee on the Ethicsof Animal Experiments in Tottori University Faculty of Medicine, and the experiments were carriedout in accordance with the Guidelines for Animal
Experiments at Tottori University Faculty of Medicine and with the Federal Law (No. 221) and Notification (No.6) of the Japanese Government.
This study comprised 2 types of experiment(see below), both on freely moving rats. All rats
were dehydrated by deprivation of drinking waterfor 24 h before experimentation. The rats lost about
6% of their total body weight as a result of this deprivation.
In Experiment 1, we investigated the effect of
an intravenous (i.v.) injection of an ACE inhibitor,lisinopril (20 mg/kg), on the LPS (2 µg/kg, i.v.)induced fever in dehydrated rats. In Experiment 2,
a single i.v. injection of lisinopril was given, and its
effect on the LPS-induced changes in the production of IL-6 in the spleen was examined, the splenic
IL-6 mRNA being measured for this purpose.

Surgery
In Experiment 1, body temperature was measured
using a biotelemetry system (Lange et al., 1991;
Data Science Inc., St Paul, MN). Each rat was anesthetized with intraperitoneally (i.p.) injected sodium
pentobarbitone (50 mg/kg) and a battery-operated
transmitter (model TA10TA-F40, Data Science)
implanted i.p. The transmitter included a sensor
and a radiofrequency transmitter. The output of thetransmitter was monitored by antennae mounted ina receiver board (model CTR86, Data Science) placed under each animal’s cage. The data were fed into
a peripheral processor (matrix model BCM100,
Data Science) connected to a Sanyo MBC-17J AX
computer (IBM compatible) (Tokyo, Japan). The
implantation of the transmitter was performed atleast 1 week before the implantation of a venous
cannula for i.v. injections.
Rats with i.p. biotelemetry transmitters wereagain anesthetized with sodium pentobarbitone (50mg/kg, i.p.) and a polyvinyl tube was inserted into
the jugular vein so that its tip lay in the superior
caval vein near the right atrium (Harms and Ojeda,

Materials and Methods

Animals
The animals used in this study were male Wistar
rats, weighing 270–350 g. They were housed inindividual plastic cages (40 × 25 × 25 cm; length ×
width × depth) with wood-chip bedding in a room
maintained at 26 ± 1˚C, a temperature within thethermoneutral zone for rats. They experienced aphotoperiod of 12 h light:12 h dark, lights coming
on at 0700. All animals had ad libitum access todrink and standard laboratory rat chow. The proto50

IL-6 and angiotensin II1974). The free end of the catheter was passed
subcutaneously to the mid-scapular region, where it
was exteriorized dorsally behind the neck. It was
kept patent by flushing it every day with heparinized 0.9% saline (50 U/mL). This implantation was
performed at least 3 days before the start of theexperiment.
In Experiment 2, the splenic expression of IL6 mRNA was measured in dehydrated rats withouttransmitters. For i.v. injections, rats were catheterized as described above.
All rats were handled for 15 min each day forat least 5 days to accustom them to the experimenters.

Experiment 2
Changes in the expression of IL-6 mRNA in the
spleen were examined in dehydrated rats following
an i.v. injection of LPS (2 µg/kg). Either lisinopril(20 mg/kg; Lisinopril + LPS group) or saline(Saline + LPS group) was administered i.v. 30 min
before the injection of LPS. The control rats received an i.v. injection of saline (vehicle for LPS) 30
min after i.v. saline (vehicle for lisinopril) (Saline +saline group). Animals were sacrificed by CO2stunning followed by decapitation either 2 or 4 hafter their 2nd injection (LPS or saline) and thespleen quickly removed, frozen and powdered inliquid nitrogen. This procedure was approved by
the Committee on the Ethics of Animal Experiments in Tottori University Faculty of Medicine.

Drugs
The LPS used in this study, which was derived from
Salmonella typhosa endotoxin (Sigma, St. Louis,MO), was dissolved in sterile saline. Lisinopril(Sigma) was also dissolved in sterile saline. The
doses injected in each experimental group are given
below.

IL-6 mRNA: The splenic expression of IL-6
mRNA was measured by Northern blot analysis. In
brief, total RNA was extracted from each tissue by
the guanidinium-thiocyanate-phenol-chloroform
method (ISOGEN; Nippon Gene, Toyama, Japan).
The RNA (20 µg) was separated, according to size,by electrophoresis on 1% agarose gels containing
6.6% formaldehyde, transferred to a nylon membrane and subjected to hybridization. The probes
were labelled with [α-32P] dCTP by the randompriming method (BcaBEST labelling kit; Takara,
Ohtsu, Japan) and purified using ProbeQuant G-50
Micro Columns (Amersham Pharmacia Biotech,
Amersham, Bucks, United Kingdom). After hybridization, the membrane was washed under stringent conditions, then subjected to autoradiography.
Images of the autoradiographs were taken into apersonal computer and analysed [Windows, Scion
Image, Plot Profile (Scion Corporation, Frederick,MD)]. The density of the IL-6 mRNA fraction,
which was normalized with respect to the β-actin
density in each sample, is expressed in arbitrary
units.
Probes for Northern blot analysis were prepared by RT-PCR. Briefly, cDNA was made fromrat spleen total RNA using an oligo(dT)17 primer bymeans of the RT reaction. Then, PCR fragments
derived from rat IL-6 and β-actin mRNA wereobtained; the fragments were of 719-bp and 762-bp,

Experimental protocols
Experiment 1
Changes in body temperature were examined inconscious rats following i.v. injection of LPS (2 µg/kg). Each rat received only 1 injection of LPS, because repeated injections result in febrile tolerance.
The injection of LPS was performed 30 min after ani.v. injection of either lisinopril (20 mg/kg;
Lisinopril + LPS group) or saline (Saline + LPS
group).
On the day of the experiment, each rat was
gently picked up and its transmitter switched onusing a magnet. The body temperature was thenallowed to stabilize for a period of 90 min before
any injections. Each injectate was given i.v. to eachanimal in a volume of 0.5 mL/kg over a period of
30 s. To minimize the influence of the rat’s own
circadian rhythm, LPS was always given between
1100 and 1200.

51

T. Watanabe et al.

respectively. The primers used for PCR were as
follows: IL-6, sense, 5'-CTTCCAGCCAGTTGC
CTTCT-3' (Siegling et al., 1994), anti-sense, 5'GCCAGTTCTTCGTAGAGAAC-3' (Bourde et al.,1996); β-actin, sense, 5'-CTATCGGCA-ATGAGC
GGTTC-3', anti-sense, 5'-CTTAGGAGTTGG
GGGTGGCT-3' (Siegling et al., 1994). Each PCR
fragment was inserted into a T vector (pT7Blue;
Novagen, Darmstadt, Germany) and cloned in E. coli
(XL1-Blue; Stratagene, La Jolla, CA). The T vectors,
which contained 1 of the above 2 PCR fragments,
were selected by sequencing. Then, the T vectors
were digested with EcoRI and XbaI and the probes
isolated.

A one-way ANOVA followed by Fisher’s
protected least significant difference test (post hoctest) was used to analyze tissue IL-6 mRNA data
(Experiment 2).
Details of the results of the various forms ofanalysis are given in the figure legends. Differences were considered significant at P < 0.05.

Results

Effect of i.v. treatment with an ACE inhibitor, lisinopril, on the LPS-induced fever under dehydrated conditions (Experiment 1)
Figure 1 shows the effect of lisinopril (20 mg/kg,
i.v.) on the LPS (2 µg/kg, i.v.)-induced fever indehydrated rats. A single i.v. injection of LPS induced a biphasic increase in body temperature inthese rats with a latency of about 90 min. This LPSinduced fever was significantly attenuated by pretreatment with lisinopril (Lisinopril + LPS groupversus Saline + LPS group).

Statistical analysis
All results are expressed as the mean ± SEM.
The body temperature data (Experiment 1)
were analyzed for statistical significance using a
repeated-measures analysis of variance (ANOVA)(Macintosh, StatView 4.0; Abacus Concepts, Inc.,
Berkeley, CA) to assess the overall effect. Analysis
was performed on data collected from the time of
drug injection onwards, i.e. from time 0.

Body temperature

(˚C)

39

38

Saline + LPS [13]Lisinopril + LPS [13]
37

0

30

60

90

120

150

180

210

240 270300

330 360

390 420 (min)Time
Fig. 1. Effect of an angiotensin-converting enzyme (ACE) inhibitor on lipopolysaccharide (LPS)-induced fever indehydrated rats. Mean values (± SEM) obtained for body temperature (˚C) in dehydrated rats after i.v. injection (attime 0) of LPS (2 µg/kg). An ACE inhibitor, lisinopril (20 mg/kg), or saline was administered i.v. 30 min before theinjection of LPS. From repeated-measures ANOVA: for treatment effect, P < 0.05; for time effect, P < 0.0001; forinteraction, P > 0.0001. [ ], number of subjects.

52IL-6 and angiotensin II

As shown in Fig. 2A, LPS induced a marked increase in IL-6 mRNA expression at both 2 and 4 hafter the injection (Saline + LPS group versus
Saline + saline group). Pretreatment with lisinopril
seemed to exert an inhibitory effect on this responseat 2 h (Fig. 2A; Lisinopril + LPS group versus
Saline + LPS group) and a semi-quantitative
analysis of the data (Fig. 2B) revealed that the effectof lisinopril was indeed statistically significant at 2
h (P < 0.05).

Effect of i.v. treatment with an ACE inhibitor, lisinopril, on the LPS-induced increasein IL-6 mRNA expression in the spleen under dehydrated conditions (Experiment 2)
Figure 2 depicts the effect of a single injection oflisinopril (20 mg/kg, i.v.) on the LPS (2 µg/kg, i.v.)induced increase in IL-6 mRNA expression in the
spleen in dehydrated rats (Northern blot analysis).

A

2h

4h

Discussion

IL-6β-actin
e

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il
pr

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li

Sa

Sa

Li

(arbitrary units)

e

S

LP

li

Sa

Sa

B

ne

il
pr

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sin

Li

P < 0.05

IL-6 mRNA

100
80
60
40
20
0
[7]

[6]
e

e

lin

Sa

+

s

ne

+

+
ril

op

sin

Li

2h

[6]

[6]
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[6]
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in
al

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[6]
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The present results showed that the LPSinduced increase in the expression of splenic
IL-6 mRNA seen in dehydrated rats was
significantly attenuated by treatment with an
ACE inhibitor, as was the fever induced by asingle i.v. injection of LPS. These results
implicate ANG II in the LPS-induced production of IL-6 in dehydrated rats. Since IL6 is thought to be a candidate for a circulating
pyrogenic cytokine (Kluger, 1991), ANG II
may contribute to the febrile response to LPS
by mediating or modulating the LPS-induced
production of IL-6. This idea is supported byour previous finding (Watanabe et al., 2000)
that although this ACE inhibitor inhibits
LPS-induced fever, it has no effect on IL-1induced fever, indicating the involvement of
ANG II in the LPS-induced production of IL1 and ultimately of IL-6. At this point, it
should be emphasized that IL-1 induces IL-6
in the tissues, and that the IL-6 then enters
the circulation to induce part of the LPSinduced fever.

4hFig. 2. Effect of an ACE inhibitor on LPS-induced increase in interleukin-6 (IL-6) mRNA expression in the spleen in
dehydrated rats. IL-6 mRNA expression in the spleen of “Saline + saline,” “Saline + LPS” and “Lisinopril + LPS”
groups. An ACE inhibitor, lisinopril (20 mg/kg), or saline was administered i.v. 30 min before a single i.v. injection(at time 0) of LPS (2 µg/kg) in dehydrated rats. A: Autoradiograms showing IL-6 mRNA and β-actin mRNA bands
in a representative animal from each group. B: Analysis of the density of IL-6 mRNA bands in each group. Meanvalues (± SEM) obtained for IL-6 mRNA in the spleen are expressed in arbitrary units. The density of the IL-6 mRNA
fraction was normalized with respect to the β-actin density for each sample. From one-way ANOVA: for treatment
effect, P < 0.0001 (at 2 h) and P < 0.01 (at 4 h) in panel B. [ ], number of subjects.

53

T. Watanabe et al.

It has recently been reported that ANG II has
proinflammatory properties. For example, previous
in vitro studies have produced results that led us to
speculate that ANG II contributes to the productionof cytokines from leucocytes stimulated with LPS(Schindler et al., 1995; Peeters et al., 1998). Interestingly, application of ANG II onto cultured mesangial cells results in the production of IL-6 in vitro(Moriyama et al., 1995). Collectively, this evidence supports the present finding that ANG II is
involved the LPS-induced production of the proinflammatory cytokine IL-6 in vivo. However, it isunknown exactly how ANG II might contribute to
the LPS-stimulated production of IL-6. One possibility is that a proinflammatory transcription factor,
nuclear factor kappa-B (NF-κB) might mediate theeffect of ANG II. In fact, it has been reported that
LPS activates NF-κB in monocytes (Muller et al.,
1993; Baeuerle and Henkel, 1994), that the expression of IL-6 is controlled at the transcriptional level
through NF-κB (Muller et al., 1993; Baeuerle andHenkel, 1994) and that ANG II activates NF-κB in
monocytes (Kranzhofer et al., 1999). Taken together, all this suggests the possibility that the activationof NF-κB by LPS is enhanced by ANG II, leading
to an increase in cytokine production. This idea
should be tested in detail in future research.
The present study has revealed at the mRNAlevel that in vivo, ANG II is involved in the LPSinduced production of IL-6, which contributes, atleast in part, to the LPS-induced febrile response in
dehydrated rats. However, it is well known thatACE not only promotes the production of ANG II,
but also inactivates bradykinin. Thus, ACE inhibitors increase bradykinin levels and so the elevatedbradykinin could conceivably have affected our results. To strengthen our hypothesis on the role of
ANG II in the production of IL-6 in response to
LPS, all the experiments performed in this study
should be repeated, using ANG-receptor antagonists, before reaching any final conclusions. On theother hand, we have previously shown an attenuation of LPS-induced fever by an ACE inhibitor not
only in dehydrated rats but also in euhydrated rats,
although the effect was greater in the former(Watanabe et al., 2000). Thus, the action of ANG II

in promoting cytokine production is not limited todehydrated conditions. It will be interesting to investigate the significance of the stimulatory actionof ANG II on the production of proinflammatory
cytokines under “euhydrated conditions,” using
relatively high doses of LPS.
Acknowledgments: We are grateful to Dr. Robert J.
Timms for his critical reading of this manuscript.
This work was partly supported by the Ministry of
Education, Science and Culture, Japan, with a Grant-inAid for Scientific Research (C12670060).

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Received March 7, 2003; accepted April 24, 2003
Corresponding author: Tatsuo Watanabe, MD, PhD

55