Induction of group II-like phospholipase A2 by lipopolysaccharide in the liver of BCG-primed rat

Vol.

174,

February

No.

3, 1991

14,

1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages

1077-l

083

INDUCTION OF GROUP II-LIKE PHOSPHOLIPASE AZ BY LIPOPOLYSACCHARIDE IN THE LIVER OF BCG-PRIMED RAT Masami Inada, Hiromasa Tojo=, Sumio Kawata, Seiichiro Tarui, and Mitsuhiro Okamotol Second

Department

, Osaka University Medical Osaka 553, Japan

"Department of Molecular Physiological Osaka University Medical School, Nakanoshima, Osaka 530, Japan

Chemistry, Received

of Internal Medicine Fukushima, Fukushima-ku,

January

4,

School,

Kita-ku,

1991

SUMMARY The specific activity of phospholipase AZ (PLA2) in the liver homogenate was elevated 1.7-fold in bacillus Calmette-Guerin (BCG)-treated rats, 1.6-fold in lipopolysaccharide (LPS)-treated rats, and 2.4-fold in BCGinfected rats treated with LPS, compared with that of control rats. These increased activities were almost completely inhibited by the antibody directed against rat splenic group II PLAz (PLA=M) but not by anti-pancreatic PLAz antibody. The results of immunoblot analysis confirmed that the PLAz immunochemically related to the group II enzyme was induced by treatment with BCG and/or LPS. The anti-PLAzM antibody-inhibitable PLAz activity per a single cell was elevated not only in nonparenchymal cell fraction but also in hepatocyte fraction, as in the case of whole liver. On the contrary, the PLAz concentration and its specific activity did not change by the same treatment both in spleen homogenate and in isolated spleen cell fractions although a 3fold increase in spleen mass occurred by BCG treatment. These results suggested that a tissue-specific mechanism of the PLA z induction by these inflammatory mediators may operate in liver. iJ 1991 Acadrmrc Press, Inc.

Phospholipase bond

at the

implicated

to play

reactions

(l-3).

classified

into

primary type

(group

(PLAzM), against (group

catalyzes

the hydrolysis

of the

of glycero-3-phospholipids.

an important

role

in

The calcium-dependent at least

structure

(4):

two groups the

the

This

pathogenesis

PLAzs

pancreatic

type

to their (group

I)

acyl

ester

has been

of inflammatory

of mammalian

according

fatty

enzyme origin

can be

characteristics

in

and the

crotalid/viperid

fractions

of rat

II).

Recently, more,

AZ (PLA2)

sn-2-position

and by using PLAzM. II-like

Abbreviations: lipopolysaccharide, factor.

we purified showed

that

a PLA2 from it

belongs

particulate

to group

PLAz category

immunochemical

techniques

we showed

PLA 2 immunochemically

PLAz) PLAz,

that

was preferentially phospholipase PLAzM: a rat

with

II

a polyclonal

distributed

related in

the

spleen

(5).

Further-

antibody

directed

to group splenic

AZ: BCG, bacillus Calmette-Guerin; splenic group II PLA,; TNF, tumor

II

enzyme

macrophages LPS, necrosis

Vol.

174,

No.

3, 1991

and Kupffer cells, are

cells thought

or immune

response.

(bacillus

Calmette-Guerin,

by subsequent wall (7,9),

In this spleen rat

processes

liver,

in rats.

not

in

parvum,

role

treatment

necrosis

in

activated

group

II

outer

vitro

factor

production

especially

bovis

(LPS)

induces,

and superoxide of PLAzs,

and then

lipopolysaccaride

such as tumor (lo),

the

phagocytic phagocytosis

of Mycobacterium

the

This

COMMUNICATIONS

namely mononuclear in host defense via

endotoxin,

and in

(TNF)

release

11).

Howev-

PLAZ,

in such

vivo.

we investigated

we obtained but

RESEARCH

by administration

(7-9).

production about

of BCG-primed

PLAZ antibody,

with responses

known study,

BIOPHYSICAL

BCG) or Corynebacterium

biological is

primed

bacteria

prostaglandin little

inflammatory

in

They are

administration

several

AND

(6). These cell populations, to play an essential role

of Gram-negative

vivo, er,

BIOCHEMICAL

the

By using evidence spleen,

effect

that

antibody

a group

by treatment

MATERIALS

of LPS on PLAz in

anti-PLAzM

with

II-like

and ant

the

PLAZ could

BCG and/or

liver

and

-pancreatic be induced

LPS.

AND METHODS

hkperimental animals: Male Sprague-Dawley rats weighing between 180-200 g were used. All animals were provided with food and water ad libitum. Animals were divided into four groups as follows: control rats, BCG alone-treated rats, LPS alone-treated rats, and BCG plus LPS-treated rats. Each group consisted of 8 rats. BCG (Mycobacterium bovis, Japan BCG Laboratory, Tokyo, Japan) was administered through a tail vein (7 X lo7 viable organisms suspended in 0.2 ml of sterile and non-pyrogenic saline per one rat). The BCG-treated animals were further treated with LPS or saline 14 days after the BCG injection when granuloma formation and the activation of the reticuloendothelial system were maximal (7). LPS prepared from E. Coli Olll:B5 (Difco Laboratories Inc., Detroit, MI) was injected via a tail vein in a dose of 250 pg in 0.25-ml saline. Animals were sacrificed 2 hours after the LPS administration under pentobarbital anesthesia. At this time the rats were in acute shock and TNF production was reported to be sufficiently stimulated; the BCG-infected rats died about 4-5 hours after the LPS treatment (7). Bloods were drawn from abdominal aorta as much as possible. Immediately, the liver was preperfused via portal vein with CaZ+-, Mg2+-free Hanks' balanced salt medium. Then, a small portion of liver and spleen tissue were removed for homogenization. Hepatocytes and nonparenchymal cells were prepared as described previously (6). Macrophages and lymphocytes from spleen tissues were also prepared as reported previously (6). Cell viability was estimated by trypan blue exclusion test and the cell numbers were determined by hemocytometer. Assay of PI& activity: Tissues of liver and spleen and pellets of isolated cells were homogenized in g-volume of 0.1 M Tris HCl buffer (pH 7.4) by using a physcotron homogenizer (Niti-on Medical and Physical Industry Co. Ltd., Chiba, Japan). PLAz activities were determined as reported previously (12,13). Fatty acids released by PLAz were derivatized with g-anthryldiazomethane, and then each derivatized fatty acid was separated by means of reverse phase high performance liquid chromatography. The assay mixture contained 5 m&l CaCIZ, 0.8 mM I-palmitoyl-Z-oleoyl-phosphatidylglycerol (Avanti Polar Inc. Co.) as a substrate, 5 m&l sodium cholate, 0.1 M NaCl, 0.1 M TrisHCl (pH 8.5). and the enzyme sample. In a control tube, CaClz was replaced by 10 mM EDTA. The PLAZ activity was expressed as nmoles of oleic acid released per min. The contribution of endogenous substrate to the measured activity was always less than about 6X. Effects of anti-PLAzM antibody on the enzyme activity were tested by the methods reported previously (6). 1078

Vol.

174,

No.

3, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

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Other analytical methods: Immunoblot analysis was carried out by the method Immunoreactive bands were described previously (14) with some modifications. visualized with a Konica immunostain HRP kit (Konica, Tokyo, Japan). Protein concentration was determined using bicichoninic acid (BCA) protein assay reagent (Pierce, IL) as recommended by the manufacturer. The data were expressed as mean value + SD and were statistically analyzed with Student's t test after the equality of variances with an F test.

RESULTS The calcium-dependent from

rats

treated

in MATERIALS liver

from

+ 1.30 (3.33

with

AND METHODS) are

+ 1.30

rats

BCG plus

LPS,

however,

of the

rats

92-942)

LPS-treated,

and BCG plus

majority

not of

related

at all the

to the

PLAz

four

(Fig.l,B).

II

in

the

PLA,

the

respective

other

spleen

the rats (7.98

treated

of treatment sum of the drug

inhibited

(5.63

activity

rats

with with

incre-

alone.

of control,

Similar

BCG-treated,

by anti-PLAzM

PLAz antibody.

On the

in

of control

those

in case

livers

were

PLA 2 was attributable enzyme.

activities

Control

the

rats

by anti-pancreatic

A

Fig.1.

LPS-treated

in

exceed the

that

rats,

than

synergistically in

as described

and LPS-treated

than

LPS-treated

with

groups

protein)

in PLAz activity treated

homogenates

The PLAz activities

elevated

of PLA2 activity

increased group

inhibitable groups

not

four

higher

In BCG plus

did

and spleen

into

Fig.1.

significantly

The increment

in activity

but

in

liver

nmol/min/mg

was significantly

proportions body,

shown

were

in

LPS (divided

+ 1.03

nmol/min/mg).

BCG or LPS alone. ments

(5.77

nmol/min/mg)

activities

BCG and/or

BCG-treated

nmol/min/mg)

+ 1.30

PLA,

This

suggests

antithat

to PLA2 immunochemically hand,

homogenates

the

anti-PLA2M

did

not

vary

antibodyamong the

**

BCG

LPS

BCGt

LPS

Control

BCG

LPS

BCGt

LPS

Effects of BCG and LPS on PLA 2 activity in rat liver (A) and spleen (B) The hatched bar represents portion of the PLAz activities homogenates. inhibited by anti-PLAnM antibody. Results were expressed as mean + SD. **; p
1079

the

Vol.

174,

No.

3, 1991

BIOCHEMICAL

C

LPS

BCG

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

BCG C

LGS

BCG

LPS

BCC Lik

Fig.2.

Immunoblot analyses with the anti-PI&M spleen (B) homogenate. Ten @g protein

The results in Fig.2. the rats

homogenates

apparently All

PLAzM.

with

from

in parallel

samples

gave

spleen

apparently

vary

with a band

homogenates,

phages

and lymphocytes

In the

isolated

by BCG and/or

liver

anti-PLAzM

isolated

and the

(Fig.4), cells,

from the

LPS administration

A

and BCG plus

same mobility

anti-pancreatic

of the

liver

the

the

four

did

not

hepatocytes were

determined.

PLA2 activities

per

cells

were

enhanced

not

nonparenchymal

cell

fraction

only

in the

-

B

*

,

**

I

L

T

I L

Fig.3.

and

monocytes/macro-

of rats lo6

LPS-treated

was observed.

namely namely

groups

in

purified

antibody

cells, cells,

shown

PLAz activias the

PLA2 antibody with

spleen

are

to increase

antibody-inhibitable

the

examined.

antibody seemed

LPS-treated,

groups

(Fig.3).

anti-PLASM antibody

immunoreactivities

among the cells

anti-PLAzM

showing

with

The PLAz activities nonparenchymal

using

BCG-treated,

No immunoreactivity

In the

analyses

Immunoreactivities

liver

ties.

of immunoblot

antibody in the liver (A) and was applied to each lane.

Control BCG LPS BCG +LPs Effects of BCG and LPS on PLAz activity nonparenchymal cells (A) and hepatocytes as mean + SD. **; p
in the isolated (B). Results

liver cells, were expressed

Vol.

174,

No.

3,

1991

BIOCHEMICAL

AND

BIOPHYSICAL

A

BCG

in

the hepatocyte

activities

of

the

fractions

ment with

liver

BCGtLPS

Control

vary

fraction

BCG

both

LPS

(6).

antibody. the

than

In all

In the

macrophage

manner

In individual

PLA 2 activity

results

in

in a similar

homogenates.

had greater

previous

ed by anti-PLAzM not

LPS

IAT

BCG+LPS

Effects of BCG and LPS on PI& activity in the isolated monocytes/macrophages (A) and lymphocytes (B). Results as mean + SD.

also

cell

COMMUNICATIONS

B

m

Fig.4.

but

RESEARCH

groups,

groups, the

spleen

fractions

to the

hepatocyte

isolated

spleen cells, were expressed

changes

non-parenchymal fractions

in

PLAz activity cells,

and in the

in PLAz agree-

was inhibit-

PLAz activities

lymphocyte

did

fractions.

DISCUSSION This

study

dothelial

demonstrated

stimulation

in

vivo

differential

by BCG and LPS on the

spleen.

The anti-PLAzM

antibody-inhibitable

enhanced

by BCG and/or

LPS administration

the PLA2 concentration of group

II-like

lial

stimulation.

been

reported

(16).

as judged

PLA2 protein Similar in

rat

and various

unknown

in

these

creased

PLAz activity. On the

the

spleen

other

liver.

in

the

into

the

Pl&

activity

treated

spleen rats.

a rat

Unlike

spleen in

isolated

mass were

in agreement per

in the

the

with did case

canine

liver

liver

(17).

spleen

increased previous increase of liver, 1081

by the

cells,

was about 3-fold infiltration results

was

(9). induction

in

synthesis

reticuloendothe-

It

have

membrane

is,

however,

for

the

in-

was observed

both

although

group

the

of control

rats

Consequently, and BCG plus per

in II

as much as that

of mononuclear

in BCG-treated the

liver

the

plasma

60 times that

and

by endotoxin

responsible

PLA2 activity

rats

appreciable

Thus,

liver

of PLA z is

the

liver

an increase

PLA2 activity

of rat

of control

indicating

the

(15),

isozyme

and in the spleen

The spleen

BCG administration,

in

fractions

no change

in with

analysis.

of enhanced

reticuloen-

PLAz in

PLA 2 activity

mitochondria which

hand,

homogenate

PLA, activity in the

reports

of the

II-like

concomitantly

was induced

subcellular

group

by immunoblot

results

liver

effects

a single

by

cells a total LPScell

Vol.

174,

did

No.

not,

known

3, 1991

BIOCHEMICAL

however,

at

occur

present.

in

the

macrophages that

rate elicited

explanation

the

isolated

and/or

liver

the

the

liver

results lation

(10).

stimulatory

action

whereas

sarily

synergistic

shown

to activate muscle

chemical

After lung

in

cell

and thymus

PLAz was,

in

the

was also

tissue

II-like enhanced

of nonparenchymal process

of the Finally,

to group role

II

splenic

each

other

different

cell

in

from

that

is

the in cell

reported

that

chemical

mediators

(21).

with

the

upon

is

stimu-

known

liver

that

(22), (23)

vivo

neces-

has been

or to enhance and rat

at present

by BCG/LPS

in

was not

TNF or interleukin-1

chondrocyte

but

in various It

the

chondrocyte

directly

number

synergistically

in

unclear

of

nonparenchymal

TNF may be involved cells

by BCG

induction

in agreement

TNF production

in

the

was enhanced

various

is

induced

Nakano

group

vascular

whether

or through

and Arita

injection

liver

both

in

study

clearly

by treatment fractions

of LPS,

control

the

in-

indirect

with

contained

action

by BCG and/or

LPS administration

study is

the nonparenchymal

The concomitant to the provides

induced

endotoxins

aorta, that the

pathogenesis

a smaller cells,

PLAz

II-like

LPS.

This

manner

of PLA2 activities

of LPS-mediated that

amount

in liver

by BCG and LPS administration. injury

extrahepatic

to that in

inflammatory

PLA zz immunochemically

and its

of

the activity

in liver

1082

II rat.

group

or BCG plus

the

spleen,

group

in a similar

elevation

evidence

but

that

of mRNA detection. relatively

than

rat

that

LPS alone

sensitivity

in

reported

and in LPS-treated

demonstrated

PLAz activity

liver. this

(25)

PLAz mRNA was enhanced

due to lower

cells.

enzyme

study,

of this

may be related

of intestinal

It

II

the

hepatocyte

cells

rat

by intravenous in

fact,

these

which

study.

in

a single

an increase

Kupffer

this

caused

may be mainly

group

is

has been

causes

vitro.

of group

results

Although

only

that

articular

of this

mRNA was undetectable

discrepancy

of

mediators.

completion

the

not

LPS on PLA2 activity

fractions

of PLA;? production in

in is

It

isolated rats

rabbit

(24)

of expression

However,

per

cell,

suggest

PLAz activity

PLAz activity

of other level

studies

as shown

in

from

cells

we concluded

produce

enhancement

PLAz mRNA level

creased

(20).

of LPS in

its

and that

of BCG and/or

by not

a single

cells,

of BCG-primed

(7,8),

smooth

be caused

by Birmelin

LPS treatment

effect

and hepatocyte

of PLAz in

Recent

by Kupffer

is

in mechanisms

different

are

this

the modifiers. have been reported:

parvum

Consequently,

Kupffer

for

difference

cells

The PLA2 activity

might

reported including

the

COMMUNICATIONS

(19).

fractions

induction

cells,

II

cells.

cell

reason

in Kupffer

uptake

the

LPS administration.

PLAz in also

of endotoxin

investigated

nonparenchymal

is

factor

macrophages

We further

RESEARCH

The precise

by Corynebacterium

by peritoneal

BIOPHYSICAL

and spleen by treatment with Kupffer cells and macrophages

of cytotoxic

The mechanism

(18).

spleen.

A possible

PLAz induction of the liver Similar differences between The production

AND

related The manifesta-

Vol.

tion

174,

No.

3, 1991

has been

insights

into

repeatedly studies

BIOCHEMICAL

AND

emphasized

(26).

on roles

played

BIOPHYSICAL

RESEARCH

The present

by PLA Z in

the

study

COMMUNICATIONS

will

pathogenesis

provide

some

of liver

disease.

A.cknsledgments: A parts of this study was supported Scientific Research from Uehara Memorial Foundation, Sankyo Life Science Foundation.

by Grants-in-Aid for CIBA-GEIGY Foundation

and

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