Muramyl dipeptide-elicited production of PGD2 from astrocytes in culture

Vol. 156, No. 2 , 1 9 8 8

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pages 882-888

October 31,1988

MURAMYL

DIPEPTIDE-ELICITED FROM

Kozo

PRODUCTION

ASTROCYTES

Y a m a m o t o I, T o s h i a k i

OF

PGD 2

IN C U L T U R E

Miwa I, Ryuji

Ueno,

and O s a m u

Hayaishi*

Hayaishi Research

B i o i n f o r m a t i o n T r a n s f e r Project, D e v e l o p m e n t C o r p o r a t i o n of Japan, c/o Osaka Medical C o l l e g e 2-7 D a i g a k u m a c h i , Takatsuki, 569, J a p a n

Received September 9, 1988

SUMMARY: We used p r i m a r y c u l t u r e s of rat b r a i n a s t r o g l i a l cells in order to i n v e s t i g a t e the i n t e r r e l a t i o n s h i p b e t w e e n PGD 2 and o t h e r s l e e p - p r o m o t i n g s u b s t a n c e s such as muramyl d i p e p t i d e (MDP), lipopolysaccharide (LPS) , d e l t a - s l e e p - i n d u c i n g peptide (DSIP) , uridine, and i n t e r l e u k i n i (IL-I). A large a m o u n t of PGD 2 was r e l e a s e d into the c u l t u r e m e d i u m by s t i m u l a t i o n w i t h MDP, LPS, and IL-I but DSIP and u r i d i n e failed to s t i m u l a t e such release. These results suggest t h a t P G D 2 m a y be p a r t of the s e r i e s of b i o c h e m i c a l steps i n v o l v e d in i n d u c t i o n of sleep by MDP, LPS, and IL-I. ® 1988 A c a d e m i c Press, Inc.

Prostaglandin mammalian such

as

central

(3) .

that

the

area

preoptic (5)

ipresent Chemical *To

whom

or

monkeys

address: Ind. Co.

(CNS)

previous

or

of

into

(6).

Besides

major plays

(I),

reports

natural the

the and

temperature

PGD 2 i n d u c e d (4)

one

system

of body

Further,

demonstrated

D 2 is

nervous

regulation

seizure

rats

(PG)

from

PGD2,

our

when

ventricle several

should

be

the roles

(2),

and

laboratory

injected of

into

conscious

putative

Biochemical Research Laboratories, Ltd., Takasago, Hyogo 676, J a p a n

correspondence

in

important

nociception

sleep

third

PG's

sleep-

Kanegafuchi

addressed.

Abbreviations used: PG, p r o s t a g l a n d i n ; MDP, muramyl dipeptide; LPS, llpopolysaccharide; DSIP, delta-sleep inducing peptide; DMEM, D u l b e c c o ' s m o d i f i e d Eagle medium; PBS, p h o s p h a t e b u f f e r e d saline; FBS, fetal b o v i n e serum; GFAP, glial f i b r i l l a r y acidic p r o t e i n ; FITC, f l u o r e s c e i n - l s o t h i o c y a n a t e 0006-291X/88 $1.50 Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

882

Vol. 156, No. 2, 1988

promoting

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

substances

lipopolysaccharide (9),

and

known

uridine

about

substances It

is

(LPS) (I0)

the

the

amounts

of

that

PGE 2

which

similarity

some

production

and

among

(MDP) peptide

However, these

with

that

PGD 2.

relationships

LPS

1

that in

of MDP In

(7) , (DSIP)

little

is

sleep-promoting

prostanoid

this

If

may have

some

PG

factor

treated

this

LPS and

with

and

MDP,

these

LPS,

(13).

with

we

MDP,

activities, is

true,

relationship

communication, culture

with

by

significant

immune-related

PGD 2.

including between

(IL-l)-like

many

produced

treated

released

astrocytes

in p r i m a r y

substances

main

astrocytes

including

activities of

the

substances,

be e x p e c t e d

astrocytes

sleep-promoting possible

described.

interleukin

prostanoids

sleep-promoting

of

PGD 2 is

(12)

and

it w o u l d

treatment

been

sleep-promoting

Therefore

the

dipeptide

(8), d e l t a - s l e e p - i n d u c i n g

have

in c u l t u r e

of

produce

muramyl

interrelationship

reported

has

as

(ii).

astrocytes one

such

the with

describe

various

the

kinds

and d i s c u s s

of the

sleep-promoting

substances.

MATERIALS

AND

METHODS

Materials: MDP and DSIP were obtained from the Peptide Institute, Osaka, Japan. LPS ( S a l m o n e l l a t y p h i m u r i u m origin) and u r i d i n e were from Sigma, St. Louis, USA. H u m a n IL-I was o b t a i n e d from G e n z y m e C o r p o r a t i o n , Boston, USA. Phosphate-buffered saline (PBS, pH 7.3) and D u l b e c c o ' s m o d i f i e d E a g l e M e d i u m (DMEM) w e r e purchased from Nissui Pharmaceuticals, Tokyo, Japan. Fetal bovine serum (FBS) c a m e f r o m G r a n d I s l a n d B i o l o g i c a l Company (GIBCO), Ohio, USA. S p e c i f i c a n t i s e r a a g a i n s t PG's were o b t a i n e d by the m e t h o d p r e v i o u s l y d e s c r i b e d (14). Glial cell c u l t u r e s : P r i m a r y glial cell c u l t u r e s w e r e p r e p a r e d a c c o r d i n g to Iwasaki et al. (15) w i t h some m o d i f i c a t i o n s . In brief, c e r e b r a l h e m i s p h e r e s f r o m n e w b o r n W i s t a r rats w e r e f r e e d from m e n i n g e s in i c e - c o l d PBS. The t i s s u e s w e r e cut into small pieces in p r e w a r m e d DMEM c u l t u r e m e d i u m w h i c h c o n t a i n e d 10% FBS, 0.05% L-glutamine, 60 U / m l penicillin G, and 74 U / m l streptomycin. The minced brain tissue was dissociated by i n c u b a t i o n w i t h 0.25% t r y p s i n for 10min. After dispersion by pipetting, the s u s p e n s l o n was f i l t e r e d t h r o u g h a lens p a p e r and washed three times by centrifugation (1000rpm, 3 min) . The

883

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

p e l l e t was r e s u s p e n d e d in the a b o v e c u l t u r e m e d i u m to w h i c h 0.13% s o d i u m b i c a r b o n a t e ( f i n a l c o n c e n t r a t i o n ) h a d b e e n added. The cell preparation was counted and seeded into 6-cm Primaria dishes (Beckton Dickinson & Co.) w i t h 5 x 1 0 5 v i a b l e cells in 5ml of c u l t u r e m e d i u m per dish. For e x a m i n a t i o n of p h e n o t y p e s , cells w e r e a l s o g r o w n on 13-mm p o l y - L - l y s i n e - c o a t e d glass coverslips. The d i s h e s w e r e m a i n t a i n e d at 37°C in a h u m i d i f i e d atmosphere c o n s i s t i n g of 90% air and 10% CO 2, and the m e d i u m w a s c h a n g e d t w i c e a week. Characterization of g l i a l c e l l s : We employed an immunocytoc h e m i c a l p r o c e d u r e u s i n g a n t i b o d y a g a i n s t glial f i b r i l l a r a c i d i c protein (GFAP) , o n e of t h e m a r k e r p r o t e i n s of a s t r o c y t e s , to characterize the cultured brain cells. The cells on the c o v e r s l i p s w e r e t r e a t e d w i t h G F A P a n t i b o d y (Dakopatts) and F I T C conjugated antisera. More t h a n 95% c e l l s w e r e p o s i t i v e a g a i n s t G F A P a n t i b o d y , w h i c h i n d i c a t e d that the c e l l s p r e p a r e d c o n s i s t e d of m a i n l y a s t r o c y t e s . T r e a t m e n t of the glial cells: PG p r o d u c t i o n i n c r e a s e d m a r k e d l y as c u l t u r e s g r e w t o w a r d c o n f l u e n c e (data not shown) as s h o w n by K e l l e r et al. (12). Since the cells b e c a m e c o n f l u e n t a f t e r 10-14 days, we c u l t i v a t e d these c e l l s for one m o n t h to get r e l i a b l e data. The s l e e p - p r o m o t i n g s u b s t a n c e s (MDP, LPS, DSIP, u r i d i n e , and IL-I) were dissolved in PBS(+)-glucose, which is P B S c o n t a i n i n g 100mg/l M g C I 2 - 6 H 2 0 , 100mg/l CaCI 2 and 1% g l u c o s e . The a s t r o c y t e s c u l t i v a t e d for one m o n t h w e r e w a s h e d three times w i t h PBS which was then replaced with prewarmed sample-containing medium. The cultured cells were incubated at 3 7 ° C in a C O 2 incubator f o r 24 h o u r s f o r t h e d o s e - d e p e n d e n c e s t u d y or f o r 0 , 3 , 6 , 1 2 , and 24 hours for the t i m e - c o u r s e study. E x t r a c t i o n and r a d i o i m m u n o a s s a y of p r o s t a g l a n d i n s : The c u l t u r e media were removed and extracted twice with two v o l u m e s of e t h y l a c e t a t e at pH 3. The PG's w e r e p a r t i a l l y p u r i f i e d w i t h a S E P - P A K C 18 c a r t r i d g e (Waters A s s o c i a t e s , Boston, MA), and PGD 2 a n d PGE 2 w e r e s e p a r a t e d by h i g h - p e r f o r m a n c e liquid c h r o m a t o g r a p h y (HPLC; C o s m o s i l C-18 column, Nakarai Chemical, Osaka). The a m o u n t s of PGD 2 and PGE 2 in the a s t r o g l i a l c u l t u r e s u p e r n a t a n t s were measured with a radioimmunoassay b a s e d on d i s p l a c e m e n t of a u t h e n t i c t r i t i a t e d d e r i v a t i v e s (15). The a s s a y was s u f f i c i e n t l y sensitive (20ng/ml) to d e t e r m i n e r e l i a b l y the a m o u n t s of t h e s e PG's. H o w e v e r , since the a m o u n t of P G F 2 ~ p r o d u c e d was too small to d e t e r m i n e under our experimental conditions, we did not m e a s u r e it.

RESULTS

Astrocytes (0.1-10~M) When

the

increase

showed

culture a

was

in

production

the

the

treated

relationship doses

of

DISCUSSION

treated

with

dose-dependent

culture

dependent between

in

AND

with of

0.1~M

and

production 10~M

PGD 2

between

10~M.

MDP,

over

MDP

884

MDP-containing

and

of

we

the

observed control.

PGD 2

While

PGD 2

formed

medium (Fig.l). a

6-fold

A

dose-

was

PGE 2 p r o d u c t i o n

seen was

VoI. 156, No. 2, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

10(~

75

== 50

2s to

C o n t . 0.1 1.0 1 0 1 0 0 MOP (pM)

0 . 0 1 0.1 1.0 10 LPS (pg/ml)

Fig. i. Dose-response of MDP- and LPS-stimulated production of PGD2(open bars) and PGE2(shaded bars).Astrocytes were incubated with PBS(+)-glucose containing MDP or LPS for 24 hours in a CO 2 incubator (37°C,5%CO2). Results are expressed as the mean+S.E.M. of PG's f o r m e d ( n g / d i s h / 2 4 h ) in 6-7 dishes. ~ : p<0.05, ~ : p<0.01 vs. the control(Student's t-test or Cochran-Cox test).

also

increased

increase

were

dependency

A

significantly less

with

the

doses

range

examined.

from

our

laboratory

report

treated a

rate

of

comparable PGD 2

to

and

(10zg/ml On

that

PGE 2 LPS)

the

uridine

of

increased

vs.

other

in

PGD 2

We

the At

a

and

the

found amount

that

However,

at

of

optimum

dose,

had

that of a

in

higher almost

production and

an

LPS-

PGD 2

PGE 2 was

LPS)

of

dose-

(10~g/ml)

also

(0.1~g/ml

no

showed

production

an

amounts

showed

LPS

large

.

12-fold

control, hand,

(i).

(Fig.l)

P G D 2.

with

produced

(10~g/ml),

(I.0pM-100~M)

elevation

formation

manner

of L P S

PGD 2

treated

in c u l t u r e

dose-dependent

concentration

slices

PGD 2

astrocytes

stimulation,

those

preoptic/hypothalamic increased

the

than

throughout

previous

by

of

33-fold

respectively.

other

sleep-promoting

and DSIP

production

substances,

(100pM-100~M),

over

the

failed

control

levels

such

as

to c a u s e

an

(data

not

shown) .

The LPS

are

time-courses shown

production

in

6 hours

of

Fig.2. after

PG

formation Both

the

in

the

compounds

initiation

885

of

presence

started

to

treatment.

of

MDP

elicit The

or PG

delay

Vol. 156, No. 2, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

,

50

i

i

B

._~ 13

40

i

~

20

O

3

6

12

24

0

3

Incubation time

6

12

24

( h )

Fig. 2. Time-dependent production of P G D 2 ( O ) and PGE2( O ) by MDP-(A) and LPS-(B) stimulated astrocytes in culture. Astrocytes were incubated with P B S ( + ) - g l u c o s e containing 100zM MDP or 10~g/ml LPS in the CO 2 incubator. Each point is the mean of 6 dishes.

in

MDP-

view

and

that

protein

Therefore,

we

inhibitors, on

PG

MDP

LPS

in M D P -

(13) , we

i).

and

inhibitors.

activation

of

RNA

(l~g/ml)

sleep

produce

the

amounts

of PG's

inhibited

reports

by

was

thought

immune

886

(izg/ml), inhibited stimulated

large

amount

astrocytes of

to

PGD 2 and

the

may

be

LPS~treated of I L - l - l i k e with

IL-I

PGE 2 r e l e a s e d

significantly

that

mediate

sustem,

Since

increased

by

synthesis of

the

mechanism

may

be

such as p h o s p h o l i p a s e ( s )

or

cell

it is s u p p o s e d

in I L - l - t r e a t e d

synthesis

synthesis.

several

enzymes

Therefore

protein

the RNA and p r o t e i n

revealed in

response.

inhibitors

(16).

a

the

the

by a s t r o c y t e s

peripheral

cultured

of P G - s y n t h e s i z i n g

induced

both

a substance

in

culture

and

in

with

cycloheximide

protein

IL-I,

production

(19).

and

Because

LPS-induced

and was

PG

cyclooxygenase m a y be also

effects

treated

Recent

IL-l-induced

involved

de novo

measured

consistent

be

the PG p r o d u c t i o n

The p r o d u c t i o n

treatment

is

might

activities and

production

i).

that

in p r i m a r y

(10units/ml)

D

requires

reported

astrocytes

(Table

the

(Table

completely,

and

involved

IL-I

examined

MDP or LPS

factor

synthesis

synthesis

It is many

PG's

actinomycin

production with

LPS-induced

astrocytes.

that

types

these

enzymes

Vol. 156, No. 2, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

T a b l e I. E f f e c t s of v a r i o u s s l e e p - i n d u c i n g production a n d its i n h i b i t i o n by R N A a n d inhibitors

Additions

s u b s t a n c e s on PG protein synthsis

n

PGD 2

PGE 2

3

7.3±1.9

1.3~0.2

3

0.5±0.03

0.4±0.03

PBS + Cycloheximide

3

1.7±0.6

0.5±0.1

MDP

3

40.4±23.0

3

4.2±1.6

0.7±0.2

3

3.1±1.2

0.3±0.04

PBS

PBS + Actinomycin

D

MDP

+ Actinomycin

D

MDP

÷ Cycloheximide

LPS

13.9~7.0

3

57.1±40

16.6±1.6

3

2.8±11

0.9±0.1

LPS + Cycloheximide

3

1.5±03

2.4±1.4

IL-I

6

32.0±85

3.1±0.9

6

0.9±03

0.3±0.1

6

0.9±04

0.4±0.1

LPS

IL-I

+ Actinomycin

D

+ Actinomycin

D

IL-I + Cycloheximide

Cultured astrocytes were i n c u b a t e d with medium(PBS) alone, MDP(100~M), LPS(10~g/ml) , or IL-l(10units/ml) in the absence or in the p r e s e n c e of the i n h i b i t o r , a c t i n o m y c i n D (l~g/ml) or cycloheximide(l~g/ml). Each value is the mean±S.E.M, of PG formed(ng/dish/24h) .

These promote may

be

also an

sleep

in the

by

important

that

induction synthesis

brain.

mechanisms. suggests

cells

suggest

through

mediated

enzymes other

results

The

While

glial

role

in

LPS,

of

PGD 2 and

of

IL-I

of

modulation

at

DSIP of

in

these

part,

responses

PG-synthesizing

may

P G D 2 in

P G D 2 in of

least

that

and/or

and

synthesis

formation

the

and

uridine

active

that

MDP

the

induce the brain

sleep-related

sleep

by

astrocytes may

have

neuronal

(20).

ACKNOWLEDGMENTS: We t h a n k Dr. H. A s o u of T o h o U n i v . a n d Dr. A O g u r a of M i t s u b i s h i - K a s e i Institute for Life Sciences for useful advise about the primary culture technique; and Ono Pharmaceuticals, Osaka, for the supply of prostaglandin standards.

887

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

REFERENCES

I.

2. 3. 4. 5. 6.

7. 8. 9. i0. ii. 12. 13. 14. 15. 16. 17. 19. 20.

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