2
.
9
. Miscellaneous Data were expressed as mean 9 S.D. Differences be-
tween groups were compared for statistical significance using the Student’s t-test. A probability value less than
5 was considered significant. The experimental proto- col was approved by the Human Ethics Review Com-
mittee and the Ethics Review Committee for Animal Experimentation of the institution.
3. Results
3
.
1
. Thioglycollate-elicited macrophages express group-II PLA
2
Various inflammatory
bioactive molecules
are secreted from cells in atherosclerotic lesions [1]. Among
them, group-II PLA
2
as a candidate for enhancing the Ox-LDL-induced macrophage growth was focused on
for the following reasons: i previous studies reported the presence of group-II PLA
2
in human atherosclerotic lesions [33,34], which played an enhancing role in
mouse atherosclerotic lesions [21,22]; and ii the pres- ence of group-II PLA
2
in peritoneal exudate induced by various stimuli [35 – 37]. RT-PCR analysis showed a
faint band of mouse group-II PLA
2
mRNA in resident macrophages, whereas a clear band of mouse group-II
PLA
2
mRNA was present in thioglycollate-elicited macrophages Fig. 1A. Next PLA
2
activity in the macrophage conditioned medium was examined. Fig.
1B shows a significantly high PLA
2
activity in the conditioned medium containing thioglycollate-elicited
macrophages compared
to that
of resident
macrophages. These results suggested that thioglycol- late-elicited
macrophages produced
a significantly
higher amount of group-II PLA
2
into the medium than resident macrophages.
3
.
2
. Ox-LDL stimulates growth of thioglycollate-elicited macrophages
The effects of Ox-LDL on the growth of mouse resident
macrophages and
thioglycollate-elicited macrophages were examined. Fig. 2A shows that
thymidine incorporation into resident macrophages was induced by Ox-LDL in a dose-dependent manner, but
neither by LDL nor acetyl-LDL. Fig. 2B shows that Ox-LDL also induced thymidine incorporation into
thioglycollate-elicited macrophages, which was three times higher than that of resident macrophages Fig. 2.
As a control, thymidine incorporation induced by 1 nM of GM-CSF was almost equal in both types of
macrophages Fig. 2. Cell-counting assay also showed
Fig. 1. Polymerase chain reaction PCR analysis of mouse group-II phospholipase A
2
PLA
2
mRNA A and PLA
2
activity in medium B. A Mouse peritoneal macrophages 2 × 10
6
cells were seeded in 3.5 cm dish and incubated for 90 min. Non-adherent cells were removed by washing with medium A. The cell monolayers thus formed were incubated in 2 ml of medium A alone for 1 h. After incubation, total RNA was extracted
from each dish with TRIzol. The expression of mRNA for mouse group-II PLA
2
upper panel or b-actin lower panel was evaluated by RT-PCR as described in Section 2. B Mouse peritoneal resident macrophages or thioglycollate-elicited macrophages 1 × 10
7
cells were seeded in 10 cm dish and incubated for 90 min. After washing with medium A, the cell monolayers thus formed were incubated at 37°C for 18 h in 10
ml of RPMI 1640 with 3 bovine serum albumin BSA. After incubation, the conditioned medium was collected and PLA
2
activity was determined as described under Section 2. After washing with PBS, cells were lysed with 0.1 N NaOH and protein contents were determined using
BCA reagent Pierce. Data represent the mean 9 S.D. of three separate experiments. P B 0.01 compared to medium alone by Student’s t-test.
Fig. 2. Oxidized low density lipoprotein Ox-LDL-induced growth of resident macrophages A and thioglycollate-elicited macrophages
B. Mouse peritoneal macrophages 4 × 10
4
cells were dispersed in each well, and incubated for 90 min. Non-adherent cells were re-
moved by washing with medium A. The cell monolayers thus formed were incubated for 6 days in 1 ml of medium A with the indicated
concentrations of Ox-LDL , acetyl-LDL or LDL
. As a control experiment, cells were incubated with 1 nM of recombinant
granulocytemacrophage colony-stimulating factor GM-CSF. Eigh- teen hours before the termination of experiments, cells were chased
with [
3
H]thymidine, harvested and radioactivity was determined as described under Section 2. Data represent the mean 9 S.D. of four
separate experiments. P B 0.01 compared to medium alone, † P B 0.005 compared to medium alone by Student’s t-test.
Fig. 3. Effect of oxidized low density lipoprotein Ox-LDL on [
3
H]thymidine incorporation into macrophages derived from human group-II phospholipase A
2
PLA
2
transgenic mice. Mouse resident peritoneal macrophages from human group-II PLA
2
transgenic mice or their negative littermates 4 × 10
4
cells were dispersed in 24 well plates, and incubated for 90 min. After washing, cell monolayers thus
formed were incubated at 37°C for 6 days in 1 ml of medium A with the indicated concentrations of Ox-LDL , acetyl-LDL , LDL
or 1 nM of recombinant mouse granulocytemacrophage colony- stimulating factor GM-CSF as a control. Thymidine incorporation
was determined as described in Section 2. Data represent the mean 9 SD of three separate experiments. P B 0.01 compared to medium
alone by Student’s t-test.
ulating activity of Ox-LDL was significantly greater than that of resident macrophages.
3
.
3
. Group-II PLA
2
enhances macrophage growth To determine the role of group-II PLA
2
in the Ox- LDL-induced macrophage growth, the effect of Ox-
LDL on the growth of macrophages obtained from human group-II PLA
2
transgenic mice was examined. Fig. 3 shows that the Ox-LDL-induced thymidine in-
that the number of resident macrophages increased 1.8 times by Ox-LDL compared to non-loaded resident
macrophages, whereas the number of thioglycollate- elicited macrophages was significantly increased 2.3
times by Ox-LDL Table 1. These results demonstrated that the growth of thioglycollate-elicited macrophages
was also induced by Ox-LDL and the responsiveness of thioglycollate-elicited macrophages to the growth-stim-
Table 1 Ox-LDL-induced growth of mouse resident and thioglycollate-elicited inflammatory macrophages
a
Macrophages Lipoproteins
Cell number ×10
− 4
well Medium alone
100 Resident macrophages
1.4 9 0.2 Medium alone
100 1.4 9 0.3
LDL 107
1.5 9 0.3 Acetyl-LDL
179 Ox-LDL
2.5 9 0.2 GM-CSF
2.8 9 0.4 200
Medium alone Elicited macrophages
1.5 9 0.2 100
107 LDL
1.6 9 0.4 Acetyl-LDL
1.8 9 0.3 127
227 3.4 9 0.2
Ox-LDL 3.0 9 0.3
GM-CSF 200
a
Peritoneal macrophages 2×10
4
cells were dispersed into culture plates, and incubated for 90 min. After incubation, non-adherent cells were removed by triplicate washing with medium A. At the start of experiment, cell numbers of resident and elicited macrophages were 1.4 and 1.5×10
4
cellswell, respectively. Macrophage monolayers thus formed were incubated for 7 days in 1 ml of medium A with 20 mgml of lipoproteins or 1 nM of GM-CSF as a control. After incubation, counting of solubilized nuclei was performed as described under Section 2. Data are expressed
as mean 9 S.D. of quadriplicate counts. LDL, low density lipoprotein; Ox-LDL, oxidized low density lipoprotein; GM-CSF, granulocyte macrophage colony-stimulating factor.
PB0.01, compared to medium alone. PB0.01, compared to medium alone by Student’s t-test.
corporation into macrophages from human group-II PLA
2
transgenic mice was twice that from their nega- tive
littermates. Moreover,
the Ox-LDL-induced
thymidine incorporation into macrophages from human group-II PLA
2
transgenic mice was significantly but partially inhibited by 50 by a polyclonal anti-human
group-II PLA
2
antibody as compared to that by non- immune IgG Fig. 4. These results suggested that
group-II PLA
2
might enhance, at least in part, the Ox-LDL-induced macrophage growth.
3
.
4
. Ox-LDL augments GM-CSF release It has recently been reported that GM-CSF played a
priming role in the Ox-LDL-induced macrophage growth [13]. Thus, the production of GM-CSF from
thioglycollate-elicited macrophages was next examined. When resident macrophages were incubated with
medium alone for 6 h, GM-CSF was released at con- centration of 8.2 fMmg protein Table 2. Ox-LDL
significantly enhanced the GM-CSF release from resi- dent macrophages into the medium in a dose-dependent
manner with a maximal concentration of 30.2 fMmg protein at 40 mgml of Ox-LDL Table 2. On the other
hand, when thioglycollate-elicited macrophages were incubated with medium alone, the concentration of
GM-CSF in the medium was 7.8 fMmg protein Table 2. However, the GM-CSF release from thioglycollate-
elicited macrophages was increased by Ox-LDL with maximal concentration of 40.5 fMmg protein, which
was significantly higher than that from resident macrophages induced by Ox-LDL Table 2. In con-
trast, LDL or acetyl-LDL could not enhance the GM- CSF release from both types of macrophages Table 2.
Combined with the results of macrophage growth Fig. 2 and PLA
2
activity Fig. 1, these results suggested that PLA
2
might enhance the effect of Ox-LDL on the GM-CSF release as well as macrophage growth. To
confirm this conclusion, the effect of Ox-LDL on the GM-CSF release from human group-II PLA
2
trans- genic macrophages was examined. As shown in Table 3,
the Ox-LDL-induced
GM-CSF release
from macrophages obtained from wild type littermates was
2.1 times greater than that from wild cells which were incubated
with medium
alone. Consistent
with macrophage growth Fig. 4, the Ox-LDL-induced
GM-CSF release from human group-II PLA
2
trans- genic macrophages was significantly greater than that
from wild macrophages Table 3. These results sug- gested that group-II PLA
2
enhances GM-CSF release from thioglycollate-elicited macrophages, thereby en-
hancing the Ox-LDL-induced macrophage growth.
4. Discussion
