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
COMMUNICATIONS
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
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
COMMUNICATIONS
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
