ATVB/2003/3582-R1 Online Supplement
Methods
Experimental animals
1,2 h-apoA-IV/E0transgenic mice were obtained as described previously . Ten-week-old male h- apoA-IV/E0 (n=16), apoE0 (n=16), and wild-type C57Bl/6 (n=10) mice were randomly divided into two equal groups. The first group were injected intraperitoneally with LPS
(Sigma, St Louis, MO, USA, 50 µg) (LPS group) and the second group with PBS (PBS group). The injections were performed once per week for 10 weeks for LPS and for 11 weeks for PBS. Animals were bled 24 h after the 1st, 4th, and 8th injections under fasting conditions.
At the end of the study animals were killed as reported previously 3. Animals were handled and cared in accordance with the Pasteur Institute Guidelines for Husbandry of Laboratory
Mice.
Plasma lipid and h-apoA-IV quantification
The plasma concentrations of cholesterol, triglycerides, and phospholipids were determined enzymatically using commercial kits (Biomérieux, Marcy l’Etoile, France). h-apoA-IV was measured by an antibody-sandwich ELISA 4.
Determination of lecithin:cholesterol acyltransferase activity and cholesterol efflux
LCAT activity was determined by use of the exogenous proteoliposome substrate method 5.
Cellular cholesterol efflux was determined using Fu5AH rat hepatoma cells incubated with
5% diluted serum as previously described 6. Purified human HDL samples were included in each assay as positive controls. All efflux values presented are averages of three determinations performed in two to four pools of plasma from each group of animals.
Evaluation of aortic lesions
Because the extent of lesions in the aortic root and the entire aorta show a significant
5 correlation in apoE0 mice , fatty streak lesions were quantified by evaluating cross-sectional lesion sizes in the aortic sinus 3.
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Determination of titers of autoantibodies to oxidized LDL
Autoantibodies directed against oxidized LDL (anti-oxLDL) were detected as reported previously 3. Goat anti-mouse horseradish peroxidase (HRP)-conjugated IgM, IgG, IgG1,
IgG2a, IgG2b, IgG3 and IgA (1:1000 dilution, Caltag Laboratories, Burlingame, CA, USA) were used. The HRP activity was determined using o-phenylenediamine dihydrochloride
(Sigma) as a substrate and detected at 492 nm. The titers of total IgM and IgG autoantibodies were determined using goat anti-mouse IgG+IgA+IgM (H+L) as a capture antibody and HRP- conjugated goat anti-mouse IgM and IgG as detecting antibodies, respectively.
Cell preparation and cytokine detection assays
Peripheral blood, spleen, thymic, and hepatic lymphocytes were isolated from each animal as previously described 3. Flow cytometry was performed as previously published 3.
Immunohistochemistry
Aortic sinus sections were fixed in acetone and blocked with FCS diluted in solution A (TBS
0.05 M + 0.05% Tween 20 + 2% BSA) (1:5). h-apoA-IV was detected by use of a biotin- conjugated anti-h-apoA-IV antibody 1. IgM and IgG were detected by use of HRP-conjugated anti-IgM (1:40, Caltag Laboratories) and HRP-conjugated anti-IgG (1:400, Caltag
Laboratories) antibodies respectively. Natural killer (NK) or natural killer T (NK-T) cells were detected by use of a FITC-conjugated anti-NK1.1 monoclonal antibody (1:30) (BDh
Pharmigen, Boston, MA, USA). IL-4 was detected by use of a PE-conjugated anti-IL-4 monoclonal antibody (1:30) (BD Pharmigen). Sections were incubated with the appropriated antibody for 1 h at 37°C and washed three times with solution A. For h-apoA-IV staining, streptavidin-conjugated alkaline phosphatase (Dako, Trappes, France, 1:300) was applied for
1 h at 37°C. Sections were incubated with Fast Red TR/Naphtol AS-MX (Sigma) or 3-amino-
9-ethyl-carbazole (AEC, Sigma) to detect alkaline phosphatase or peroxidase, respectively, and counterstained with Mayer’s hematoxylin solution. Endogenous alkaline phosphatase and
2 ATVB/2003/3582-R1 Online Supplement peroxidase activities were blocked with levamisole (Sigma) and hydrogen peroxide, respectively. Controls were performed with an irrelevant isotype-matched monoclonal antibody, and no significant background staining was visualized.
Monocyte cell separation and TNF- production
Human peripheral blood monocyte cells were isolated by Ficoll-Hypaque density gradient purification and high magnetic gradient sorting using the MIDIMACS technique (Miltenyi
Biotec Gmbh, Bergisch Gladbach, Germany), as described previously 8. The resulting preparations were 99.6 % pure. Monocytes were incubated overnight at 37°C, in a 10% CO2 atmosphere, in round-bottom microtiter wells (105 cells/well) in RPMI medium with 10%
FCS containing 2 µg/ml brefeldin A (Sigma) in different stimulation conditions: LPS, recombinant h-apoA-IV, and a recombinant h-apoA-IV/LPS equimolar mix pre-incubated in medium for 1h at 37°C. Monocytes were also pre-incubated with h-apoA-IV for 4h at 37°C, in a 10% CO2 atmosphere and washed two times, before being stimulated with either LPS or the h-apoA-IV/LPS mix. Unstimulated monocytes (incubated in PBS) were used as a baseline control. After overnight incubation at 37°C in a 10% CO2 atmosphere, the production of TNF-
by monocytes was assessed by intracellular staining with saturating amounts of anti-human
TNF- monoclonal antibody (BD Pharmingen). Similar experiments were performed with apoA-I purified from human plasma (h-apoA-I) (Sigma).
Statistical analysis
Data are expressed as mean ± standard deviation (SD). Statistical analyses were performed using the Statview 4.1 software (Abacus Concept Inc). Differences between groups were analyzed by one-way ANOVA and were considered to be significant if P<0.05.
References
1. Ostos MA, Conconi M, Vergnes L, Baroukh N, Ribalta J, Girona J, Caillaud JM, Ochoa
A, Zakin MM. Antioxidative and antiatherosclerotic effects of human apolipoprotein A-
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IV in apolipoprotein E-deficient mice. Arterioscler Thromb Vasc Biol. 2001;21:1023-
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human apolipoprotein AIV gene expression in transgenic mice. FEBS Lett. 1999;445:45-
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3. Ostos MA, Recalde D, Zakin MM, Scott-Algara D. Implication of natural killer T cells in
atherosclerosis development during a LPS-induced chronic inflammation. FEBS Lett.
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4. Ostos MA, Recalde D, Baroukh N, Callejo A, Rouis M, Castro G, Zakin MM. Fructose
intake increases hyperlipidemia and modifies apolipoprotein expression in apolipoprotein
AI-CIII-AIV transgenic mice. J Nutr. 2002;132:918-923.
5. Chen C, Albers JJ. Characterization of proteoliposomes containing apoprotein A-I: a new
substrate for the measurement of lecithin: cholesterol acyltransferase activity. J Lipid Res.
1982;23:680-691.
6. de la Llera Moya M, Atger V, Paul JL, Fournier N, Moatti N, Giral P, Friday KE,
Rothblat G. A cell culture system for screening human serum for ability to promote
cellular cholesterol efflux. Relations between serum components and efflux, esterification,
and transfer. Arterioscler Thromb. 1994;14:1056-1065.
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correlation between lesions in the aortic origin and in the entire aorta, and differences in
the extent of lesions between sexes in LDL receptor-deficient and apolipoprotein E-
deficient mice. J Lipid Res. 1995;36:2320-2328.
8. Miltenyi S, Muller W, Weichel W, Radbruch A. High gradient magnetic cell separation
with MACS. Cytometry. 1990;11:231-238.
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Figure Legends
Figure I. Immunohistochemical study of representative aortic sinus sections from LPS- injected apoE0 (A and C) and h-apoA-IV/E0 (B and D) mice. Natural killer (NK) or natural killer T (NK-T) cells were detected by use of a FITC-conjugated anti-NK1.1 monoclonal antibody (A-B). IL-4 was detected by use of a PE-conjugated anti-IL-4 monoclonal antibody
(C-D). Original magnification x400.
Figure II. TNF- production by human monocytes. Purified monocytes (A) were incubated with LPS (B), with recombinant h-apoA-IV protein (C), and with a preincubated equimolar mix of LPS and h-apoA-IV (D). Additionally, monocytes were preincubated with h-apoA-IV before stimulation with LPS (E), or with an equimolar mix of LPS and h-apoA-IV (F). TNF- production was detected by intracellular staining (shaded curves). The unshaded curves represent isotype controls. The results shown are representative of four independent experiments.
Figure III. TNF- production by human monocytes. Purified monocytes (A) were incubated with LPS (B), with apoA-I purified from human plasma (h-apoA-I) (C), and with a preincubated equimolar mix of LPS and h-apoA-I (D). Additionally, monocytes were preincubated with h-apoA-I before stimulation with LPS (E), or with an equimolar mix of
LPS and h-apoA-I (F). TNF- production was detected by intracellular staining (shaded curves). The unshaded curves represent isotype controls. The results shown are representative of two independent experiments.
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