Vol. 171, No. 2, 1990 September

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RESEARCH COMMUNICATIONS Pages

14, 1990

coIvvuL!3ANTs

INDUCB

Masabumi

INTERLEUMN-1s

Minami’,

* Department

l

Yasushi Kuraishi’, Takashi Yamaguchi”, and Masamichi Satoh*+

of Pharmacology, Faculty of Pharmaceutical Kyoto University, Kyoto 606, Japan

Sciences,

* Cellular Technology Institute, Otsuka Pharmaceutical Tokushima 771-01, Japan

August

1,

832-837

MESSENGEIR RNA IN RAT BRAIN

Yoshikatsu Hirai”,

Satoru Nakai”,

Received

AND BIOPHYSICAL

Co.,

1990

SUMMARY: The effects of systemic administration of kainic acid and pentylenetetrazol on interleukin-l/? gene expression in the rat brain was studied. After the administration of kainic acid in a convulsive dose (10 mg/kg i.p.), Interleukin-l/3 mRNA was induced intensely in the cerebral cortex, thalamus and hypothalamus, moderately in the hippocampus and weakly in the striatum, but not in the midbrain, pons-medulla and cerebellum. Pentylenetetrazol induced Interleukin-l/? mRNA in the cerebral cortex, hypothalamus, and hippocampus with a faster time-course than kainic acid. Diazepam suppressed both the convulsion and the induction of Interleukin-$3 mRNA produced by kainic acid. Dexamethasone suppressed the induction of Interleukin-lp mRNA, but did neither the convulsion nor the induction of c-fos mRNA following the injection of kainic acid. These results provide the first evidence that intensive neuronal excitation induces Interleukin-l/? mRNA in particular regions of the brain. ml990 Academic Press,Inc. Interleukin-1

(IL-l)

is a cytokine that has multiple biological activities involved in

the immune and inflammatory

responses (1). &l-like

binding sites (5) were found in the brain.

immunoreactivities

A direct administration

of IL-l

induces various actions, such as induction of sleep (6), loss of appetite hormones (8) and analgesia (9). Most recently, Interleukin-lp to inhibit long-term slices (10).

potentiation

of the mossy fiber-CA3

These findings suggest that IL-l

lipopolysaccharide

(LPS) (ll),

(IL-l/?) was demonstrated

system in mouse hippocampal

in the brain,

it

there.

is

unclear

Although

whether

changes

should be addressed.

Inc. reserved.

832

in

To estimate this issue, the

Abbreviations: IL-l, interleukin-1; IL-la, interleukin-la; NGF, nerve growth factor; LPS, lipopolysaccharide. 0006-291X/90 $1.50 Copyright 0 1990 by Academic Press, All rights of reproduction in any form

(7), secretion of

acts on neurons in the brain.

activity could alter the synthesis of IL1

’ To whom correspondence

into the brain

physical lesion (12) and several cytokines (13) have been

shown to increase the synthesis of IL1 neuronal

(2,3,4) and IL1

ILl#l,

interleukin-l/l;

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influences of convulsion evoked by drugs on the level of mRNA protein of IL,-l/? (and that of ILla

in some experiments)

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coding the precursor

in various regions of the brain

were examined in the rat. Dexamethasone

is known to suppress the synthesis of IL-Q3 mRNA in promonocytic

cell line U-937 (14) and glial cell line (1.5) in vitro.

Therefore,

we also investigated the

effects of this steroid on the induction of IL-l/3 mRNA in the brain and on the induction of c-fos mRNA, which was expressed following convulsion (16), as well. MATERIALS

AND METHODS

Materials: Male Sprague-Dawley rats (6-7 weeks old) were used. Kainic acid and pentylenetetrazol were purchased from Nacalai Tesque (Kyoto, Japan) and Sigma (St. Louis, U.S.A.), respectively. Diazepam and dexamethasone 21-phosphate disodium were gifts front\Yamanouchi Pharmaceutical Co. (Tokyo, Japan) and Takeda Chem. Ind. (Osaka, Japan), respectively. Rat IL-l/3 cDNA was cloned into lgtl0 (ll), and subcloned into pGEM (Promega, U.S.A.). Rat IL-la cDNA was cloned into pcDE (13). The probe for v-fos (PstI-PvuII fragment, lkb) was purchased from Takara (Kyoto, Japan). Treatment and tissue preparation: Rats were intraperitoneally injected with kainic acid or pentylenetetrazol (both dissolved in saline) and killed after various times. Dexamethasone (dissolved in saline) or diazepam (suspended in 0.5% carboxymethylcellulose saline) was intraperitoneally injected 10 or 5 min, respectively, before the injection of kainic acid. The brain was rapidly removed and dissected into eight regions, that is the cerebral cortex, hippocampus, striatum, thalamus, hypothalamus, midbrain, pons-medulla and cerebellum. Each tissue was rapidly frozen in liquid nitrogen and stored at -80°C until use. Tissues of each region from 2 or 3 animals were pooled for the extraction of total RNA. Isolation. fractionation and hvbridization of RNA: Total RNA was extracted from each sample according to the methods of MacDonald et al. (17). RNA samples (20 or 30 pg each) were fractionated by electrophoresis on 1% agarose gel containing 6% formaldehyde. RNA was transferred to nylon membrane (Biodyne, Pall, U.S.A.), and baked at 80°C for 2 h. The membrane was prehybridized and then hybridized to a radiolabeled cDNA or cRNA probe. Hybridization was performed at 42 or 63°C for cDNA or cRNA probe, respectively. Radiolabeling of cDNA probe with [a3’P]-dCTP (110 TBq/mmol, Amersham, U.K.) or cRNA probe with [a32P]-CTP (15 TBq/mmol, Amersham, U.K.) was carried out using a Random Primer DNA Labeling Kit (Takara, Japan) or Riboprobe Gemini System II (Promega, U.S.A.), respectively. The membrane was washed twice in 2xSSC containing 0.1% SDS for 5 min, and then twice in O.lxSSC containing 0.1% SDS for 30 min at 50 or 68°C for cDNA or cRNA probe, respectively. The membrane was exposed to X-ray film at -80°C using intensifying screen. Autoradiograms were scanned by a densitometer (CS-9000, Shimadzu, Japan) for quantitation. RESULTS An injection of kainic acid (10 mg/kg, i.p.) produced abnormal behavior in rats; “wet dog shakes”, a preconvulsive

symptom, appeared at 0.5-l h and tonic-clonic 833

convulsion

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brake out 2-2.5 h after the injection.

AND

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After intraperitoneal

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injection of kainic acid in a

dose of 10 mg/kg, about 80 % of rats showed ‘wet dog shakes” and about 70 % showed convulsion.

In following experiments, therefore,

we used the rats that had been struck

with convulsion, except an experiment using diazepam in which all of the rats injected with kainic acid were used. We first examined the levels of IL-V

mRNA in eight brain regions 2.5 h after the

injection of kainic acid, when tonic-clonic convulsion was observed. kainic

acid intensely

induced

IL-Q!? mRNA

in the cerebral

As seen in Fig. 1,

cortex,

thalamus

and

hypothalamus, moderately in the hippocampus and weakly in the striatum, but not detected in the midbrain, ILl/?

pons-medulla

and cerebellum.

When 20 pg of total RNA was assayed,

mRNA was not detected in all eight regions of the brain in non-treated

Then, we investigated

control rats.

the detailed time-courses of expression of IL-l/3 mRNA

cerebral cortex, thalamus, hypothalamus injection of kainic acid, IL-l/l

and hippocampus

(Fig. 2).

At 1 h after the

mRNA was detected only in the hippocampus,

the cerebral cortex, thalamus, and hypothalamus

in the

but also in

at 1.5 h. The maximum levels of IL$9

mRNA were obtained at 1.5 h in the cerebral cortex, 2.5 h in the thalamus and 3.5 h in the hypothalamus,

respectively.

The increase in IL-l#l mRNA

in the hippocampus

was

12345676

- 26s - 18s

01

02

0

1

2

3

Time

after

4 injection

5

24 (hour)

IL@ mRNA expression in the brain 2.5 h after the injection of kainic acid P&J. (10 mg/kg, i.p.). Total RNA (20 pg) was blotted on each lane, and hybridized to 3zP-labeled cDNA probe. Lane 1: cerebral cortex; lane 2: hippocampus; lane 3: striatum; lane 4: thalamus; lane 5: hypothalamus; lane 6: midbrain; lane 7: pons-medulla; lane 8: cerebellum. No expression was detected in lanes 6, 7 and 8. &Q. Time courses of IL-l@ mRNA expression in several regions of the brain by kainic acid (10 mg/kg, i.p.). The relative levels of IL-16 mRNA were quantified by densitometric scanning. The values are represented as percents of cerebral cortex at 2.5 h. 0: cerebral cortex; 0: hippocampus; A: thalamus; A: hypothalamus. 834

72

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relatively small but lasted for 24 h. IL@

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mRNA was no longer detected in the all four

brain regions 72 h after the injection of kainic acid. On the other hand, IL-la mRNA was induced only in the cerebral cortex 2.5 h after the injection of kainic acid (10 mglkg, i.p.)

in a much smaller extent than ILlg Pentylenetetrazol

mRNA.

(50 mg/kg, i.p.), a well known convulsant, produced typical clonic

convulsion with peak time of 5-10 min after the injection and induced IL-l/3 mRNA in the cerebral cortex, hypothalamus after the injection.

and hippocampus, but not in the other brain regions, 30 min

IL-&9 rnRNA was no longer detected in the all three brain regions 3 h

after the injection of pentylenetetrazol. A pretreatment

with diazepam (10 mg/kg, i.p.) 5 min before the treatment of kainic

acid (10 mg/kg, i.p. for 2.5 h) suppressed both the epileptic behavior and the induction

of ILV

mRNA in the cerebral cortex, hypothalamus, thalamus, hippocampus and striatum

(Fig. 3). On the other hand, a pretreatment

of dexamethasone in a dose of 1 or 3 mg/kg,

but not 0.3 mg/kg, 10 min prior to the treatment abolished

the induction

hypothalamus pretreatment

of ILlfi

mRNA

of kainic acid (10 mg/kg, i.p. for 2 h)

in the cerebral

cortex,

(Fig. 4), but did not inhibit the epileptic behavior. with dexamethasone

hippocampus

Moreover,

and

such a

did not affect an induction of c-fos mRNA by kainic

acid in these three regions.

Cerebral

cortex

12345678910

Hypothalamus

Kainic

03

1

/. I ”

04

acid

Dexamethasone

0 10

10 10 10 (mg/kg)

0

0.3

0

1

3 (mg/kg)

F_iR.. Suppression by diazepam of the induction of lL-l/l mRNA by kainic acid. Total RNA (20 pg) was blotted on each lane, and hybridized to 32P-labeledcDNA probe. Lanes 1-5: kainic acid (10 mg/kg, i.p. for 2.5 h) alone; lanes 6-10: diazepam (10 mg/kg, i.p.) 5 min before the injection of kainic acid (10 mg/kg); lanes 1 and 6: cerebral cortex; lanes 2 and 7: hippocampus; lanes 3 and 8: striatum; lanes 4 and 9: thalamus; lanes 5 and 10: hypothalamus. m. Suppressive effect of dexamethasone on the induction of IL-l/l mRNA by kainic acid. Total RNA (30 pg) was blotted on each lane, and hybridized to 32P-labeled cRNA probe. Dexamethasone (0.3-3 mg/kg, i.p.) was administered 10 min before treatment with kainic, acid (10 mg/kg, i.p. for 2 h).

835

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DISCUSSION The present study demonstrated

that two kinds of agents producing

convulsion

induced IL-l/I mRNA in the rat brain. Kainic acid-induced expression of IL-la first observed in the hippocampus, hypothalamus.

and then in the cerebral

cortex, thalamus

Such a sequence is roughly corresponding to that of propagation

activities following the injection of kainic acid (18). Furthermore, expression of IL-l/l

gene was abolished by a pretreatment

suppressed the convulsion by kainic acid. induced

IL-l/?

mRNA

with

a shorter

Another

gene was

of seizure

the kainic acid-induced

with diazepam which also

convulsant, pentylenetetrazol,

duration.

and

These findings

also

suggest that

the

extraordinary neuronal excitation enough for a production of convulsions contributes to the induction of IL-l/? mRNA by these agents. It is unlikely that the kainic acid-induced expression of IL-la gene occurs in blood cells, because the degrees of mRNA in particular

induction were different

no induction was detected in the midbrain,

among brain regions and

pons-medulla

Cultured astrocytes (19) and microglia cells (20) produce IL-l LPS.

In addition,

the number of IL-l-like

brain from patients with Alzheimer IL-l/l-like

immunoreactivity

immunoreactive

and cerebellum.

following treatment

with

glial cells is greater in the

disease and Down syndrome (3). On the other hand,

has been shown to localize in the nerve terminals in human

(2) and rat (4) b rains. In the present study it was not determined whether cells expressing IL-+S mRNA population

after kainic acid treatment

expressing IL-la

gene after kainic acid treatment,

experiments using in situ hybridization Pathophysiological

are glias or neurons.

To identify

the cell

we are now conducting

technique.

significance of convulsant-induced

IL-Q9 gene expression is not

clear. The induction of IL-l@ mRNA by the convulsants was great in the cerebral cortex, thalamus and hypothalamus, striatum, and undetectable cerebellum.

moderate

in the hippocampus,

in the

in the caudal regions such as the midbrain, pons-medulla

Such a regional difference in the IL-lp

to the distribution

weak but definite

of cellular degeneration

and

mRNA induction is roughly parallel

after systemic injection

of kainic acid (21).

Thus, it seems that the expression of II..+3 gene following convulsion is at least in part related with cellular damage. Dexamethasone,

which was reported to inhibit IL-Q3 mRNA

synthesis in some cell lines (16,17), suppressed the kainic acid-induced expression of IL-@ gene but did not inhibit the convulsion. direct

cause to

Glucocorticoids

produce

the

Therefore,

convulsion

IL-l/? gene expression may not be a

following

the

injection

of

enhance neuronal damage produced by kainic acid (22). 836

kainic

acid.

On the other

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hand, convulsion increases mRNA of nerve growth factor (NGF) in the brain (23) and further,

IL-l/I

induces NGF in periphery

cells after nerve lesion (24).

These findings

together with our present data suggest that the suppression of IL-l/? gene expression by glucocorticoid

might be involved in its action to enhance neuronal damage.

If such is the

case, IL-Q3 might play a role in the recovery of neurons from damage by extraordinary excitation. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.

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