[11]. The role for MSR-A in plaque formation is indi- cated by studies showing that MSR-A knockout mice
fed on a high cholesterol diet have a significantly reduced development of atherosclerotic plaques [12,13].
Increased intake of oily fish or fish oils containing long chain n-3 polyunsaturated fatty acids confers pro-
tection against cardiovascular and ischaemic heart dis- ease in man [14,15]. Addition of fish oil to a diet high
in saturated fats reduced the development of atheroscle- rotic plaques in mice [16], while fish oil given orally to
Watanabe heritable hyperlipidaemic rabbits resulted in a significant reduction of aortic cholesterol content and
a lowered development of aortic atherosclerotic lesions [17].
We hypothesise that some of the anti-atherogenic effects demonstrated by dietary fish oils may be medi-
ated through effects on ICAM-1 and MSR-A expres- sion on macrophages. Therefore, in this study we
investigated the effects of diets containing 20 by weight of different oils fish oil, safflower oil, coconut
oil on surface and mRNA expression of the adhesion molecule ICAM-1 and two scavenger receptors MSR-
A types I and II by murine peritoneal macrophages, a convenient source of inflammatory macrophages.
2. Methods
2
.
1
. Animals and diets Male C57Bl6 mice Harlan-Olac, Bicester, UK were
fed on a low fat LF diet containing 25 gkg corn oil or on high fat diets containing 200 gkg coconut oil
CO, safflower oil SO or fish oil FO purchased from ICN Biomedicals, High Wycombe, UK. The high
fat diets also contained 10 gkg corn oil to prevent essential fatty acid deficiency. All diets contained identi-
cal amounts of protein 200 gkg, starch 200 gkg, sucrose 295.8 gkg and vitamin E 1.2 gkg. The fatty
acid composition of the diets is shown in Table 1. Animals were allowed free access to the diets for 12
weeks n ] 8diet, and were then killed by an overdose of CO
2
. All procedures involving animals were ap- proved by the UK Home Office under the Animals
Scientific Procedures Act 1986.
2
.
2
. Chemicals Unless otherwise stated, all chemicals were purchased
from Sigma, Poole, UK.
2
.
3
. Macrophage preparation To elicit macrophage migration to the peritoneal
cavity, 4 days prior to sacrifice the mice were injected intraperitoneally with 1 ml of Brewer’s thioglycollate
broth. After death the peritoneal exudate was collected by washing out the peritoneal cavity with 4 ml sterile
PBS Oxoid, Unipath, Basingstoke, UK. The cells were washed with sterile PBS, collected by centrifugation,
passed through lens tissue Whatman, Loughborough, UK and washed again with sterile PBS. Contaminating
erythrocytes were lysed by a 5-min incubation with Tris-buffered 0.14 mM ammonium chloride, pH 7.2.
2
.
4
. Fatty acid analysis Total lipid was extracted from macrophages with
chloroformmethanol 2:1 vv and washed twice in 0.88 KCl. Fatty acids were prepared by saponification
at 70°C in methanolic 0.5 M KOH. Samples were neutralized using concentrated sulfuric acid and fatty
acids were extracted into chloroform and washed twice in 0.88 KCl. After evaporation to dryness, fatty acid
methyl esters were prepared by reaction with an excess of diazomethane in ether. Fatty acid methyl esters
dissolved in hexane were separated by gas chromatog- raphy in a Hewlett-Packard 6890 gas chromatograph
fitted with a 25-m × 0.32-mm BPX70 capillary column, film thickness 0.25 mm. Helium at 2.0 mlmin was used
Table 1 Fatty acid composition of the diets used
a
Diet LF g100 g total fatty acid
CO g100 g total fatty acid SO g100 g total fatty acid
FO g100 g total fatty acid ND
10:0 6.8
1.6 ND
3.4 56.5
12:0 4.3
ND 14:0
3.5 17.6
1.1 10.3
13.7 22.0
16:0 8.7
7.8 ND
ND 16:1 n-7
ND 14.5
18:0 3.3
5.1 3.9
4.2 21.9
2.0 18:1 n-9
19.4 11.8
54.3 2.3
18:2 n-6 61.0
9.0 18:3 n-3
ND ND
ND 3.5
ND 20:5 n-3
ND ND
10.6 ND
22:6 n-3 ND
ND 10.1
a
The mice were fed low fat LF, coconut oil CO, safflower oil SO or fish oil FO diets. ND indicates not detected.
as the carrier gas and the splitsplitless injector was used with a split:splitless ratio of 10:1. Injector and
detector temperatures were 250 and 270°C, respectively. The column oven temperature was maintained at 170°C
for 12 min after sample injection and was programmed to then increase from 170 to 200°C at 5°Cmin before
being maintained at 200°C for 15 min. The separation was recorded with HP GC Chem Station software.
Fatty acid methyl esters were identified by comparison with standards run previously.
2
.
5
. Extraction of RNA and re6erse transcription Total RNA was extracted from 2 × 10
6
macrophages using TRIzol Life Technologies, Paisley, UK in accor-
dance with the manufacturer’s instructions. Messenger RNA was then selectively reverse transcribed using an
oligo dT primer from 4.5 mg of total RNA. Reverse transcription RT was achieved with 7.5 U of avian
myeloblastosis virus reverse transcriptase Promega, Southampton, UK in the presence of 1 mM dNTPs
Pharmacia, Milton Keynes, UK, 5 mM MgCl
2
Promega, RT buffer 10 mM Tris – HCl pH 8.8, 50 mM KCl and 0.1 Triton X-100 Promega and 0.5 mg
poly dT
15
Promega. RNA was substituted with an equal volume 5 ml diethyl pyrocarbonate DePc
treated water as a negative control. Reverse transcrip- tion was carried out for 1 h at 42°C followed by heating
at 94°C for 3 min to inactivate the enzyme. The result- ing cDNA was diluted with 15 ml of DePc treated water
to a final volume of 35 ml and used as a polymerase chain reaction PCR template.
2
.
6
. DNA amplification and 6isualisation PCR was performed for a housekeeping gene cy-
clophilin, MSR-A type I, MSR-A type II and ICAM- 1. Amplification of 2.5 ml of cDNA was achieved using
1 U of Taq polymerase in the presence of 15 pmol of primer, Mg-free buffer 19 mM Tris – HCl pH 9.0, 50
mM KCl and 0.1 Triton X-100 Promega, 1.5 mM MgCl
2
0.5 mM for ICAM-1 Promega and 0.2 mM dNTPs Pharmacia. The reaction cycling was 95°C for
30 s, 56°C 60°C for ICAM-1 for 30 s and 72°C for 1 min in a Hybaid Touchdown Thermocycler. The opti-
mised number of cycles used reflecting the exponential phase of the reaction was 26 for MRSA type I and
type II and 30 cycles for ICAM-1. The primer se- quences used for cyclophilin were 5-TTGGGTCGC-
GTCTCGTTCGA-3 sense and 5-GCCAGGACC- TGTATGCTTCA-3 antisense. Primers for MSRA
type I were 5-GGGAGACAGAGGGCTTACTGG A- 3 sense and 5-TTGTCCAAAGTGAGCTCTCTTG-3
antisense 389 bp. Primers used for MSRA type II were 5-GGGAGACAGAGGGCTTACTGGA-3 sense
and 5-ATGTTCAGGGAGTTATACTGATC-3 anti- sense 223 bp. Primers used for ICAM-1 were 5-
TTTTGCTCTGCCGCTCTGGAG-3 sense and 5-TA- CACATTCCTGGTGACATTC-3 antisense 287 bp.
PCR products were electrophoresed on 2 agarose gels stained with ethidium bromide. The resultant
bands were visualised with an UV transilluminator and the image stored with a GDS 5000 gel documentation
system UVP, Cambridge, UK. The images were then analysed by densitometry using Phoretix 2D 4.00 soft-
ware Phoretix International, Newcastle-upon-Tyne, UK. All results are expressed as a ratio of ICAM-1 or
scavenger receptor: cyclophilin mRNA where the test cDNA was amplified for cyclophilin concurrently with
amplification for the ICAM-1 or scavenger receptors and under the same conditions.
2
.
7
. Receptor expression The macrophages were washed twice using modified
PBS with 0.1 wv bovine serum albumin and 10 mM sodium azide. Aliquots of 2 × 10
5
cells were incubated with
antibody against
murine monocytes
and macrophages F480, murine MSR-A type I + II 2F8
or murine ICAM-1 KAT-1 all from Serotec, Oxford, UK for 20 min at 4°C. The cells were washed twice
again with the modified PBS and then collected by centrifugation. Cells were incubated with a fluorescently
labelled second antibody STAR-49 Serotec, Oxford, UK for 20 min at 4°C. The cells were washed as before
and fixed with 200 ml of PBS containing 2 vv formaldehyde. The cells were analysed using a FAC-
Scan flow cytometer BectonDickinson, Oxford, UK. Results are expressed as percent of cells positive for
each receptor and as median fluorescence to give an indication of level of receptor expression per cell. The
expression index has been calculated as proportion of cells positive for each receptor multiplied by the median
fluorescence intensity to reflect overall changes in level of receptor expression within the cell population.
2
.
8
. Sca6enger receptor function MSR-A function was assessed by modified LDL
uptake. Acetylated LDL Ac-LDL was chosen as a well characterised modified LDL for which MSR-A
mediates 80 of uptake by macrophages [18]. Optimal conditions for uptake of Ac-LDL by freshly isolated
peritoneal macrophages were established by two prelim- inary experiments using fluorescently 1,1-dioctadecyl-
1-3,3,3,3-tetramethyl-indo-carbocyanine
perchlorate labelled Ac-LDL Biogenesis, Bournemouth, UK.
These investigated uptake of labelled Ac-LDL over time and with increasing concentrations of unlabelled
Ac-LDL Biogenesis, Bournemouth, UK in a competi- tive binding assay.
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
