Table 2 Fatty acid composition of murine peritoneal macrophages
a
CO g100 g total fatty acid SO g100 g total fatty acid
LF g100 g total fatty acid FO g100 g total fatty acid
Fatty acid diet 3.4 9 0.4
c
14:0 0.7 9 0.4
b
0.5 9 0.5
b
1.9 9 1.0
bc
21.9 9 1.1
bc
21.0 9 0.9
bc
22.4 9 1.1
c
19.5 9 0.3
b
16:0 3.3 9 0.9
c
0.0 9 0.0
b
16:1 n-7 2.6 9 0.2
c
1.9 9 0.6
c
0.3 9 0.3 0.4 9 0.4
0.2 9 0.2 0.0 9 0.0
17:0 18.9 9 1.9
18:0 18.3 9 0.6
16.7 9 0.6 16.8 9 0.3
15.8 9 0.7
d
11.7 9 0.2
c
16.2 9 0.6
cd
7.9 9 0.4
b
18:1 n-9 4.8 9 1.2
2.4 9 0.2 18:1 n-7
3.9 9 0.1 4.2 9 0.9
10.3 9 0.4
c
24.7 9 0.3
e
13.1 9 0.3
d
6.9 9 0.3
b
18:2 n-6 0.0 9 0.0
0.8 9 0.8 20:2 n-6
0.0 9 0.0 0.0 9 0.0
1.2 9 0.5
cd
2.1 9 0.2
d
0.9 9 0.4
bc
0.0 9 0.0
b
20:3 n-6 19.0 9 0.9
c
17.5 9 1.1
c
20:4 n-6 8.7 9 0.4
b
22.0 9 1.0
d
0.0 9 0.0
b
0.0 9 0.0
b
0.0 9 0.0
b
11.7 9 0.6
c
20:5 n-3 22:0
0.0 9 0.0 0.0 9 0.0
0.0 9 0.0 0.3 9 0.3
0.2 9 0.2
b
0.0 9 0.0
b
0.3 9 0.3
b
10.7 9 0.3
c
22:5 n-3 0.9 9 0.7
b
22:6 n-3 0.6 9 0.6
b
0.0 9 0.0
b
9.3 9 0.2
c
24:0 0.8 9 0.5
0.3 9 0.3 0.4 9 0.4
0.0 9 0.0
a
Mice were fed a low fat LF, coconut oil CO, safflower oil SO or fish oil FO diet. All values are means 9 S.E. for n = 3–6 mice. Values across a row not sharing a common alphabetical superscript are significantly different ANOVA, PB0.05
Freshly isolated macrophages were washed twice with PBS. Aliquots of 2 × 10
5
cells were incubated with fluorescently labelled Ac-LDL for 3 h at 37°C. The cells
were washed twice again with PBS and fixed in PBS with 2 vv formaldehyde. Uptake of fluorescently
labelled Ac-LDL was measured by flow cytometry as described above. Results are expressed as percent of
cells positive for Ac-LDL uptake and as median fluorescence to give an indication of level of uptake of
Ac-LDL per cell. The uptake index has been calculated as proportion of cells positive for Ac-LDL multiplied
by the median fluorescence intensity to reflect overall changes in level of Ac-LDL uptake within the cell
population.
2
.
9
. Data analysis All data are expressed as means 9 S.E. of n observa-
tions. Data were analysed using a one-way ANOVA with a post-hoc least significant difference test using
SPSS version 6.1 for Windows SPSS, Chicago, USA. A value of P B 0.05 was taken to indicate statistical
significance.
3. Results
3
.
1
. Yield and purity of macrophages Cell yields were 19 – 36 × 10
6
per animal. The cells were \ 85 macrophages as assessed by flow cytome-
try. There were no statistical differences in cell yield or purity between the diets.
3
.
2
. Fatty acid composition of macrophages Macrophages from the CO fed mice had a signifi-
cantly higher proportion of 14:0 when compared with macrophages from the mice fed on the LF and SO diets
Table 2. Macrophages from mice fed on the FO diet had a significantly lower proportion of 18:1 n-9 com-
pared with those from mice fed on each of the other diets. Macrophages from mice fed on the FO diet had
significantly lower proportions of the long-chain n-6 fatty acids 18:2 n-6, 20:3 n-6 and 20:4 n-6 when com-
pared with those from mice fed on each of the other high fat diets. Linoleic acid 18:2 n-6 and arachidonic
acid 20:4 n-6 were also found at significantly lower levels in the macrophages from FO fed mice compared
with the LF fed mice. Conversely the long chain n-3 polyunsaturated fatty acids 20:5 n-3, 22:5 n-3 and 22:6
n-3 were found in significantly higher proportions in the macrophages from the FO fed mice compared with
those from the mice fed on each of the other diets.
3
.
3
. Receptor expression The percentage of macrophages expressing ICAM-1
was lowest for mice fed the FO diet Table 3. The median fluorescence intensity, which indicates level of
receptor expression per macrophage, was also lowest for cells from the mice fed the FO diet. The ICAM-1
expression index was lowest for the macrophages from mice fed the FO diet and this was significantly lower
than that seen for macrophages from mice fed the CO diet Table 3.
The percent of macrophages expressing MSR-A type I + II varied from 60 to 80 with macrophages from
Table 3 ICAM-1 and MSR-A type I+II expression by murine peritoneal macrophages
a
Diet MSR-A type I+II
ICAM-1 MFI
Expression index Positive
MFI Expression index
Positive 74.0 9 4.8
63.8 9 3.2
bc
LF 68.6 9 6.8
bc
87.1 9 3.0 74.1 9 12.2
54.5 9 14.0
bc
89.4 9 6.5 77.3 9 6.6
c
CO 79.3 9 2.8
c
86.7 9 3.9 74.1 9 8.4
58.3 9 6.3
c
78.4 9 6.0 70.9 9 6.6
bc
73.6 9 2.9
c
89.7 9 2.6 76.4 9 9.9
SO 56.7 9 8.1
c
FO 79.7 9 5.0
73.3 9 5.0 59.7 9 6.5
b
59.0 9 4.6
b
54.0 9 1.9 32.0 9 3.0
b a
Mice were fed on a low fat LF, coconut oil CO, safflower oil SO or fish oil FO diet. Results are means 9 S.E. for percentage positive positive, median fluorescence intensity MFI and expression index for n = 8–12 mice. Values in a column not sharing a common alphabetical
superscript are significantly different ANOVA, PB0.05.
mice fed on the FO diet having the lowest values Table 3. A significantly lower proportion of macrophages
from mice fed on the FO diet expressed MSR-A type I + II compared to those from mice fed on the CO or
SO diets. The median fluorescence intensity, which indicates level of receptor expression per macrophage,
was also lowest for cells from the mice fed the FO diet. The MSR-A type I + II expression index was again
lowest for mice fed on the FO diet and this was significantly lower than the MSR-A type I + II index
for cells from mice fed the CO or SO diets Table 3.
3
.
4
. Macrophage sca6enger receptor function Uptake of Ac-LDL was lowest for the macrophages
from mice fed the FO diet both by percentage of macrophages positive for Ac-LDL and median fluores-
cence intensity Table 4. The uptake index for Ac-LDL was significantly lower for macrophages from mice fed
on the FO diet compared with those from mice fed on the SO diet.
3
.
5
. ICAM-
1
and macrophage sca6enger receptor mRNA
Results are shown as the ratio of ICAM-1 or MSR-A mRNA to the constitutively expressed cyclophilin
mRNA as assessed by densitometry Fig. 1A,B,C. Messenger RNA specific for ICAM-1 was significantly
lower in macrophages from the FO fed mice compared with those from mice fed on the LF or CO diets Fig.
1A. ICAM-1 mRNA in macrophages from the FO fed mice was also lower than that from mice fed the SO
diet. Freshly isolated macrophages from mice fed on the SO or FO-rich diets had less mRNA specific for
MSR-A type I than macrophages from mice fed on CO or LF diet but this difference was not statistically
significant. Fig. 1B. Macrophages from mice fed on the FO diet had significantly less mRNA for MSR-A
type II than those from mice fed on the LF diet Fig. 1C. This was also lower than that found in cells from
mice fed on the other high fat diets although this did not reach significance. Messenger RNA for MSR-A
type II was not altered by feeding on the high fat diets containing CO and SO when compared with feeding on
the LF diet Fig. 1C.
4. Discussion
