Effects of the polysaccharide chain of lipopolysaccharide in an experimental massive hepatic cell necrosis model

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1305-1310

Vol. 155, No. 3, 1988 September 30, 1988

EFFECTS OF THE POLYSACCHARIDE CHAIN OF LIPOPOLYSACCHARIDE IN AN EXPERIMENTAL MASSIVE HEPATIC CELL NECROSIS MODEL

Y.Mizoguchi, 1Y.Sakagami, 1H.Kuboi, 1K.Kobayashi, 1 and I.Yano 2 iThe Third

Department of Internal Medicine, 2Department of Bacteriology, Osaka City University Medical School, Osaka, Japan

Received August

17, 1988

SUMMARY : When small amounts (l~g) of lipopolysaccharide (LPS) purified from Salmonella minnesota (SM) wild, SM R60 and SM R345 were intravenously injected into mice 7 days after heat-killed Propionibacterium acnes was intravenously injected, massive hepatic cell necrosis was induced and most of the mice died within 24 hours. However, when LPS from SM R345 treated galactosidase, SM R5 and SM R7 and lipid A from SM R595 were administered, the survival rate was much higher and no histological changes of the liver such as necrosis could be seen in any of the mice. In each of the LPS used in this study, the structure of the polysaccharide chain was different and it was shorter in the following order: S M w i l d ÷ S M R 6 0 + S M R 3 4 5 + S M R345 treated with g a l a c t o s i d a s e ÷ SM R 5 ÷ SM R7-> SM R595. This suggests that the polysaccharide chain of LPS plays an important roie in the induction of massive hepatic cell necrosis in this experimental model, e 1988 AcademicPress, Inc.

Macrophages infiltrate into inflammatory areas in chronic inflammatory diseases

and

are

inflammation. induced

not

(MAF)

have by

to

play

cell-to-cell

activated

produced

vivo

this

induced

by factors

from

liver

massive

an

important

demonstrated

and lipopolysaccharide

factors in

We only

macrophages

thought

the

contact

(LPS)

injury

cell

vitro

in

that

between

the

liver

target

such as the macrophage

induction

macrophages

due

to

necrosis

(2).

activated

in

mice

injury

is

liver

cells

and

activating

In order

rats

by

factor

factor or to

macrophages,

and

of

cell

(i), but also by a cytotoxic

activated

cell

hepatic

in

role

study

we

have

intravenously

injecting heat-killed Propionibacterium acnes (P. acnes) and a small amount of

a

gram-negative

experimental by

the

injection

injection necrosis a

of

of

LPS

within

two-step

first

model,

the

(3).

secreting

the

by

massive

P.

a week's

acnes,

the

Most

of

after

the

acnes

cytotoxic

at

cells

24 hours

by

LPS,

mononuclear

P.

activation,

primed

which

endotoxin,

the

and

cell

animals

cells

second (3).

(3). into

die

In the

activated

of massive

(3).

activated

is

are

by

the

the cell

that by are

producing

and

LPS,

in

by

macrophages

to elucidate

induced

this liver

hepatic

It is thought

including

In order

necrosis

interval

accumulated

macrophages

LPS injection

adherent

factor

hepatic

and

are

this

the mechanism experimental

0006-291X/88 $1.50 1305

Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

V o l . 155, N o. 3, 1988

model,

various

BIOCHEMICAL A ND BIOPHYSICAL RESEARCH C O M M U N I C A T I O N S

kinds

of

LPS

with

different

polysaccharide

chains

were

examined. MATERIALS AND METHODS i. Materials. Male BALB/c mice (6 to 8 weeks old) were purchased from Keari (Osaka, Japan). P. acnes was donated by the Department of Bacteriology, Osaka City University Medical School. LPS purified from SM wild, SM R60, SM R345, SM R5 and SM R7 and lipid A purified from SM R595 were obtained from LBL Co. Their structures are shown in Fig.l. 2. Preparation of LPS from SM R345 treated with galactosidase LPS from SM R345 was incubated with galactosidase (i000 units/ml, Sigma Co.) at 37°C for 30 minutes. After incubation, this solution was heated at 100°C for 2 minutes. As the control, LPS was incubated without galactosidase. The structure is also shown in Fig.l

Salmonella minnesota Wild

I GIcNAc I

Gat

Hep

I

I

I I

8

8• IIII

O-antigen ,-{--GIc--GaI--GIc--Hep--Hep--II--~--

I

I

GIcNAc

I;

Gal Hep

I

I

T•

.•

~

I~

/

I

GIc-j-,'GaI--GIc--Hep--Hep--~

R6o

,

I

Gel R345

I

Hep

~

r--t---t---

I

•

8

I

--1--

I

•

lipid A

I

I

GIc--Hep--Hep--m---~--

;

Rs

I

Hep

lipid A

/

Gald-GIc--Hep--Hep--IIl--•----I---

R345 treated with galactosidase

lipid A

I

/

•I

•I

I:

I

lipid A

Glc-~/,Hep--Hep--'•--ll-" lipidA l

R7

Hep--Hep~II--~

lipid A

I I

R595

m--i-- ripid A GIc GIcN Gal Hep

Figure

i

: Glucose : Glucosamine : Galactose : L glycero-D-mannoheptose

• : OCH2 CH2 NH2 • : 2-keto-3-deoxy-octonate • : Phosphoric acid

The structures of LPS from Salmonella minnesota wild, Salmonella minnesota R60, Salmonella minnesota R345, Salmonella minnesota R345 treated with galactosidase, Salmonella minnesota R5 and Salmonella minnesota R7 and lipid A from Salmonella minnesota R595.

1306

Vol. 155, No. 3, 1988

BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS

3. Induction of massive hepatic cell necrosis One milligram of heat-killed P. acnes suspended in 0.i ml of saline solution was intravenously injected into each mouse through a tail vein. Seven days later 1 g of LPS from S M wild, SM R60, or SM R345, SM R345 t r e a t e d w i t h g a l a c t o s i d a s e , SM R5 or SM R7 or lipid A from SM R595, each of which was suspended in 0.I ml of saline solution containing 0.2% triethylamine, was intravenously injected, and massive hepatic cell necrosis was induced. The survival rate 24 hours after the injection of LPS was evaluated. 4. Histological studies The liver tissue was removed from the mice still alive 24 hours after LPS injection and was examined microscopically after hematoxylin-eosin staining.

RESULTS l.Effects

of v a r i o u s

LPS

cell

necrosis

was

from

S_MM wild,

S M R60

LPS

injection

w h e n LPS

and

from

induced

2. E f f e c t s

acnes

and

the

S M R345

A from S M R595 w e r e

tissue

the

in

survival

injected, LPS

mice

in

extent

in

liver

from

the all

liver of

mice.

with

mice

mice

less

survival

P.

than

30%.

noted

Fig.3

massive

and

shows

hepatic

the

hand,

of liver

was

histological necrosis

100%

(Fig.

When

the

injected

2). liver

with

90-

80

**

-....

. . . . .

......

i!iiii:

-:.:-; -.-.-.

,:.;.:. .., . . .

-...-, ....-,

1:18 :i:!:i: i:i:!: -X-;

.....

.,.,...

,., ,.,

,.,

......

.-.

-X':

:'X':

:';-:':

.....

70. "~ L 60 =

",>

:-:-X

.*~ ~

50,

;;:;:.:

40-

:::::::

30

i::i::::::::

... :.X."

seen

to

Changes

24 hours

a

large of

the

after

the

Wild

R60

R845R34~, +treated

Figure 2

R5

:':':':

....... :':':';

R7

:'":",

lipidA

with galactosidase

Effects of LPS from Salmonella minnesota wild, Salmonella minnesota R60, Salmonella minnesota R345, Salmonella minnesota R345 treated with galactosidase, Salmonella minnesota R5 and Salmonella minnesota R7 and lipid A from Salmonella minnesota R595 on survival rate of mice. ~ ~';p< 0.01

1307

P.

histologically,

%

100

LPS

On the other

necrosis

necrosis

cell

then

after

SM R60 and SM R345 was examined were

and

S M R5 and S M R7 and lipid

changes cell

hepatic

as an hour

rate was n e a r l y

hepatic

massive

acnes

as early

galactosidase,

the

When

injecting

occurred

rate was with

of

by

death

massive

cells

the

a mouse

rate

on h i s t o l o g i c a l

with

and LPS from S M wild,

changes

the

S M R345,

treated

of v a r i o u s

from

on survival

Vol. 155, No. 3, 1988

Figure 3

injection mice

Histological changes of liver obtained from a mouse 24 hours after the injection of LPS from Salmonella minnesota wild. LPS from Salmonella minnesota wild was intravenously injected 7 days after P. aches was intravenously injected, and massive hepatic cell necrosis was induced (HE stain, xlO0).

of

LPS

injected

dase, into

SM R5 the

and

P.

SM R7

macrophages,

and

be

in all

seen

from

with

lobules

liver

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

from

only

Figure 4

acnes and

17 the

and

lipid

However, LPS

necrosis, mice.

with

Fig.

spotty

and not

when

from

A from

lymphocytes

spotty

a mouse

LPS f r o m SM R345

SM wild.

SM R345

SM R595 plasma

massive

4 shows

necrosis

the

liver

tissue

treated

was

with

examined,

cells hepatic

was cell

hours

after

infiltration

necrosis, changes

the

in

the

could of the

injection

treated with galactosidase.

Histological changes of liver obtained from a mouse 24 hours after the injection of LPS from Salmonella minnesota R345 treated with galactosidase. LPS from Salmonella minnesota R345 treated with galactosidase was intravenously injected 7 days after P. acnes was intravenously injected, and only spotty necrosis was induced (HE stain, xlO0).

1308

the

galactosi-

observed

the h i s t o l o g i c a l 24

from

of

Vol. 155, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

DISCUSSION Ferluga by

and

successive

minimal

dose

necrosis

is

injected

into

Allison

(4) have

intravenous of

LPS.

induced

injections

We

have

when

mice

and

induced of

also

(3).

hepatic

injury

Corynebacterium

reported

heat-killed

rats

lethal

that

P.acnes

and

Furthermore,

parvum

massive LPS

and

hepatic

are

using

in mice a

cell

intravenously

this

experimental

model, we have shown that prostaglandin (PG) E 1 increases the survival rate of mice and remarkably improves the histological PGE I

not

only

suppresses

inhibits

the

the

release

of

activation

the

cytotoxic

of

changes of the liver (5).

liver

factor,

adherent but

it

cells

also

and

directly

affects the liver cells and protects them from the effects of the cytotoxic factor

(5).

analyze

In the present

the

different

pathogenesis

polysaccharide

the polysaccharide

study,

of

we

applied

this

massive

hepatic

experimental

cell

model

necrosis,

chains and lipid A were used,

LPS

to

with

and the effects

of

chain on the induction of massive hepatic cell necrosis

were evaluated. LPS

generally

consists

p o l y s a c c h a r i d e region side

chain which

region which has

been

binds with

indicated

endotoxin

can

is near

of

be

a polysaccharide

divided

into

the nonreducing lipid

that

A and

various

the

and

lipid

A.

The

O-antigenic

polysaccharide

end and the core

polysaccharide

is near

the

biological

are due to lipid A, but

chain

reducing

activities

its O-specific

end

(6,7).

It

of

LPS

an

as

antigenicity is confined

to the polysaccharide chain (8-10). In

this

study,

injected

with

LPS

injected

with

LPS

massive

from

SM wild,

from R345

and lipid A from S M R595. chain was broken, SM

R 6 0 ÷ SM

This

suggested

necrosis. in. the

cell

SM

treated

R60 with

necrosis and

SM

was

R345,

induced but

galactosidase,

not

in

mice

in

those

SM R5 and

SM R7

In each of the LPS, a part of the polysaccharide

and its structure was shorter in the order of SM w i l d ÷

R 3 4 5 + R345

garactosidase

hepatic

treated

with

galactosidase÷ SM R 5 ÷ S M

that the polysaccharide and

SM R345

affected

the

chain between induction

R7÷SM

SM R345

R595.

treated with

of massive

hepatic

cell

That is, the structure of the polysaccharide chain was important activation

of

macrophages

infiltrated

into

the

liver

by

the

injection of P. acnes and in the production of the cytotoxic factor. By changes

the

so-called

S ÷R

change

into the rough type,

in

which

the O-antigenic

the

smooth

polysaccharide

LPS of the S type is removed along with its toxicity activation, alternate These

(ii).

Salmonella

side chain of In complement

it is known that the polysaccharide chain of LPS activated the

pathway

facts

type

and

indicate

lipid A alone.

that that

lipid the

LPS receptors

A activates

various

activities

are thought

1309

the

classical of

LPS

to be present

pathway

are

not

(12). due

to

on the surface of

Vol. 155, No..3, !.988

macrophages some

effect

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(13), and the structure of the polysaccharide chain may have on its

binding

with

these

receptors.

In any

case,

it is

suggested that t h e polysaccharide chain of LPS plays an important role in the induction of massive hepatic cell necrosis in this experimental model.

i. 2. 3. 4. 5. 6. 7. 8. 9. i0. ii. 12. 13.

REFERENCES Mizoguchi, Y., Shiba, T., Ohnishi, F., et al. (1981) Hepato-gastroenterol. 28, 250-253. Mizoguchi, Y., Shiba, T., Ohnishi, F., et al. (1981) Hepato-gastroenterol. 28, 250-257. Tsutsui, H., Mizoguchi, Y., Yamamoto, S., et al. (1986) Cells of the hepatic sinusoid, p.307. The Kupffer Cell Foundation, The Netherland. Ferluga, J., Allison, A.C. (1978) Lancet If, 610-611. Mizoguchi, Y., Tsutsui, H., Miyajima, K., et al. (1987) Hepatology. 7, 1184-1188. Jann, K. and Jann, B. (1977) Surface carbohydrates of the procaryotic cell, p.247. Academic Press, New York. Wilkinson, S.G. (1977) Surface carbohydrates of the procaryotic cell, p.97. Academic Press, New York. Hellerqvist, C.G, Lindberg, B., Svensson, S., et al. (1968) Carbohydr. Res. 8, 43-55. Hellerqvist, C.G., Lindberg, B., Svensson, S., et al. (1969) Acta Chem. Scand. 23, 1585-1590. Hellerqvist, C.G., Lindberg, B., Samuelson, K., et al. (1971) Acta Chem. Scand. 25, 955-961 Luderitz, O., Freudenberg, M.A., Galanos, C., et al. (1982) Curret Topics in Membranes and Transport 17, 79-151. Morrison, D.C. and Kline, L.F. (1977) J. Immunol. 118, 362-368. Haeffner-Cavaillon, N., Chaby, R., Cavaillon, J.M. et al. (1982) J. Immunol. 128, 1950-1954.

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