Extract
Note: Please read the complete
full text with Figures and Tables at
Introduction
CpG-oligodeoxynucleotides (CpG-ODN), the synthetic
oligodeoxynucleotides containing unmethylated CpG
dinucleotides in specific sequence contexts, are novel
immune enhancers for systemic and mucosal
immunization[1]. Animal studies suggest that CpG-ODN could be used as
vaccine adjuvants, or antiallergenic, chemotherapeutic, or
immunoprotective agents[2-5]. Although the immunological
effects of CpG-ODN have been widely
studied[6-9], including the mechanism by which CpG-ODN binds to the cell surface
and the way in which its immunostimulatory activity is
modulated by extracellular pH[10], its biological activities are not
comprehensively understood. To improve our knowledge
of the biological activities of CpG-ODN, we used a DNA
microarray to profile the gene expression in an immune cell
line after CpG-ODN treatment, and found that some genes
related to the formation of macrophage foam cells were
upregulated. We therefore reasoned that CpG-ODN might
affect the lipid metabolism and the formation of foam cells.
Foam cells are characteristic pathological cells in
atherosclerotic lesion. An excess lipid loading in macrophages results
in the formation of foam cells, and this is a key process in the
early stages of atherosclerotic
lesions[11]. Therefore, it is very important to investigate how CpG-ODN affects the
formation of foam cells. In the present study, we investigated
the effects of CpG-ODN on the gene expression of the
related receptors or enzymes involved in atherosclerosis, and
further confirmed the results by using semiquantitative
RT-PCR. Furthermore, by histochemical and high performance
liquid chromatography (HPLC) analysis, we demonstrated
that CpG-ODN could affect the formation of foam cells.
Materials and methods
CpG-ODN ODN-1826 (5¡¯-TCC ATG ACG TTC CTG ACG
TT-3¡¯) and ODN-2006 (5¡¯-TCG TCG TTT TGT CGT TTT GTC
GTT-3¡¯) were synthesized by Sangon Biotechnology Co in
Shanghai, China.
Cell culture The mouse macrophage line RAW264.7 and
the human monocyte line U937 were obtained from the Cell
Bank in the Shanghai Institutes of Biochemistry and Cell
Biology, Chinese Academy of Sciences, and were cultured
in RPMI-1640 medium (Gibco/BRL, Rockville, USA),
supplemented with 10% fetal bovine serum, 2 mmol/L glutamine,
and 50 mg/mL gentamycin in 5% CO2 at 37
oC. The culture medium was changed every 48 h.
Analysis of gene expression profile of immune cells
by using a cDNA microarray The MGEC-80s microarray
obtained from Biostar Genechip (Shanghai) was used.
RAW264.7
cells were stimulated with ODN-1826 6 mmol/L for 6 h, and
total RNA was isolated from cultured cells using TRIzol
reagent (Gibco). mRNA was purified using the Oligotex mRNA
midi Kit (Qiagen, Germany). The fluorescent cDNA probes
were synthesized from total RNA by reverse transcription
and then purified, referring to the protocol of
Schena et al and Shalon D et
al[12,13]. The probes from the control were
labeled with Cy3-dUTP; those from the stimulated cells by
ODN-1826 were labeled with Cy5-dUTP. The probes were
mixed and precipitated with ethanol, and resolved in 20 µL
hybridization solution [5×SSC+0.4% sodium dodecylsulfate
(SDS)+50% formamide+5× Denhardt¡¯s solution]. Chips were
prehybridized with the hybridization solution+3 µL
denatured salmon sperm DNA at 42 oC for 6 h. After denaturing at
95 oC for 5 min, the probe mixture was added to
the prehybridized chip and covered with glass. Hybridization
was performed at 60 oC for 16 h. After hybridization of a
sample to the microarray, the sample was removed, and
arrays were washed in solutions of 2×SSC + 0.2% SDS, 0.1%
SSC + 0.2% SDS, and 0.1×SSC at 60
oC, respectively, for 10 min each, then dried at room temperature. Arrays were
scanned with a laser scanner (ScanArray4000, General
Scanning Inc, Watertown, Massachusetts) at 2 wavelengths. The
acquired images were processed using a modification of
GenePix Pro 3.0 software. The intensity of each spot at the 2
wavelengths represented the quantity of Cy3-dUTP and
Cy5-dUTP, respectively. Each ratio of Cy3 to Cy5 was computed.
The overall intensity was normalized by a coefficient
according to the ratios of the 40 housekeeping genes that were
also located.
Semiquantitative RT-PCR Total RNA of the U937 cells
was extracted, purified using the Tissue/Cell Total RNA
Isolation Kit (Watson, Shanghai), and quantified spectro-
photometrically. A total of 200 ng RNA was reverse
transcribed to cDNA in a volume of 20 µL using oligo(dT)
primers and a commercially available kit (Reverse Transcription
System, Promega, USA) according to the manufacturer¡¯s
instructions. For transcription, the RNA was denatured at
70 oC and then transcribed for 1 h at 42
oC. After inactivation of the avian myeloblastosis virus (AMV), (1 min at 99
oC), the transcript was cooled down to 4
oC. Four microliters of the reaction mixture was subjected to a final PCR reaction in
a total volume of 20 µL, containing 0.5 µL of
Taq polymerase (Takara Biotechnology, Dalian, China), 2 µL of 10×PCR Buffer
(Mg2+ Plus, Takara, Dalian), 2 µL dNTP mix (2.5 mmol/L), 0.5
µL of each of the 5¡¯ and 3¡¯ primers, and 10.5 µL of
H2O. All gene-specific PCR primers were designed based on the DNA
sequences in GenBank (National Center for
Biotechnology Information, Bethesda, MD, USA). The sequence of the
primers and the annealing temperatures are shown in Table
1. The amplification was carried out over 35 cycles of the
following: denaturation, 1 min at 94 oC; annealing, 30 s, at
temperature see Table 1; elongation, 1 min at 72
oC. PCR products were separated by agarose gel electrophoresis.
b-actin was used as a control. The intensities of the
gene-specific PCR products were quantified from scanned images
using the AlphaEase FC software (Alpha).
Preparation of ox-LDL Low-density lipoprotein (LDL)
(d=1.019 to 1.063 kg/L; Sigma) was sterilized by filtration
through 0.45 µm Millipore membranes, and stored at 4
oC as described
previously[14]. After edetic acid was removed by
dialysis, LDL were oxidized by incubation in
CuSO4 10 µmol/L at 37
oC for 12 h, and then dialyzed in phosphate buffered
saline (PBS) containing edetic acid 0.1 mmol/L at 4
oC for 24 h.
In vitro induction of foam cell formation
Confluent U937 cells were pretreated in serum-free RPMI-1640 for 24 h and
then incubated with ox-LDL 80 mg/L for 48 h. The foam cell
model was thus established. In the experiments, U937 cells
were cultured with ODN-2006 6 µmol/L and ox-LDL 80 mg/L
for 48 h, which were added to the medium simultaneously.
The control group was the U937 cells without ODN-2006
treatment. The morphological form of the foam cells was
analyzed by histochemical methods.
Histochemical analysis of foam cells The foam cells
were collected and fixed with 4% paraformaldehyde for 12 h
on the slides as described
previously[15]. The slides were rinsed in water and placed in fresh 0.3% oil red O (Amresco,
Solon, USA) solution for 20 min. The slides were rinsed in
50% isopropyl alcohol and examined.
Lipid extraction Lipids were extracted by using the
method of Hara and Radin[16] with modifications. The cells
were collected from the culture flasks into 0.9% NaCl (2 mL
per 75 cm2 flask) and homogenized on ice by sonication for
10 s with a Sonifier 450 sonicator (Branson Ultrasonics,
Danbury, USA) set to maximum power. The protein
concentration of the cell lysate was determined by the method of
Lowry et al[17]. To a volume of cell suspension known to
contain 1 mg of protein was added 100 µg of cholesteryl
heptadecanoate in chloroform as an internal standard. An
equal volume of freshly prepared cold (-20
oC) KOH in ethanol (150 g/L) was then added and the cell lysate was
repeatedly vortexed until clear with 6% trichloroacetic acid. An
equal volume of 4:1 hexane-isopropanol
(v/v) was added and the mixture was vortexed for 5 min followed by
centrifugation at 800×g and 15
oC for a further 5 min. The
extraction procedure was repeated twice (a total of three extraction
procedures). The combined organic phase was transferred
to clean tapered glass tubes and thoroughly dried in a
vacuum freeze dryer at 65 oC. The tubes were allowed to cool
to room temperature, 100 µL of the mixture of isopropyl
alcohol-n-heptane-acetonitrile at 35:12:52
(v:v:v) was added, and the sample was solubilized by placing it in an ultrasound
water bath for 5 min at room temperature. After
centrifugation at 800×g for 5 min, 20 µL of the sample was introduced
into the HPLC device.
Determination of cholesterol and cholesteryl ester
content of U937 cells by HPLC The cholesterol and cholesteryl
ester content of cells were analyzed by HPLC as described
previously[18]. HPLC was performed using a Waters device
(Milford, USA) equipped with a model 1525 binary pump, a
model 717 plus autosampler, a model 2487 dual l absorbance
detector, and a 4.6 mm×100 mm Gen-Pak FAX column
(Waters). Waters¡¯ Breeze software was used to control the
HPLC system. Cholesterol and cholesteryl esters were eluted
isocratically at a flow rate of 0.5 mL/min and at a temperature
of 4 oC using an eluent consisting of
isopropanol-n-heptane-acetonitrile at the ratio of 35:12:52
(v:v:v) and detected by ultraviolet absorption at 206 nm.
Statistics Every experiment was repeated at least 3 times.
All values are expressed as mean±SD. Differences were
considered statistically significant when P<0.05 as determined
by the paired t-test.
Results
Gene expression profile in immune cells By using the
cDNA microarray, 119 genes related to cell cycle, immune
modulation, and signal transduction were found to be
differentially expressed, of which 3 genes related to the formation
of macrophage foam cells had increased expression after
ODN-1826 6 mmol/L stimulation (Table 2).
Confirmation of foam cell-related gene expression by
RT-PCR According to the 3 genes in Table 2, we designed
primers and performed semiquantitative RT-PCR. The
expression of these genes in the ODN-2006 treated group was
markedly increased, compared with the control group (Figure 1).
Effect of CpG-ODN on lipid deposition in cells
Lipid drops inside the foam cells were stained with oil red O, and
some of the red pellets were found in the plasma of the U937
cells after being incubated with ox-LDL, indicating that the
foam cell model was formed. After ODN-2006 6 mmol/L was
added to the medium, lipid deposition in U937 foam cells was
enhanced (Figure 2).
Effect of CpG-ODN on cholesterol and cholesteryl ester
in foam cells After incubation with ODN-2006 6
mmol/L and ox-LDL 80 mg/L for 48 h, the level of cholesteryl ester in foam
cells increased markedly compared with the controls (Figure
3). This suggests that CpG-ODN can also increase lipid
accumulation (based on the assay of the concentrations of
cholesteryl ester and the ratio of cholesteryl ester to total
cholesterol; Table 3). Cholesterol concentration has a strong
linear relationship with its peak area within 0.05-1.0 mg/L
(r2 in each case was >0.998).
Discussion
Previous reports have suggested that CpG-ODN is mainly
involved in immunomodulation[19,20]. Nevertheless, in the
study presented here, we investigated immune cells treated
with CpG-ODN using cDNA microarray technology and,
surprisingly, found that the expression of 3 genes (CD36
[NM-007643], LPL [NM-008509], and Fcg2b [NM-010187])
increased markedly. These results were further confirmed
by semiquantitative RT-PCR. CD36, LPL, and Fcg2b are
thought to be closely related to lipid metabolism and the
formation of macrophage foam cells, therefore CpG-ODN is
able to modulate gene expression in foam cells, and increase
lipid accumulation in foam cells. Our findings suggest that
CpG-ODN has a novel biological activity.
Atherosclerosis, and the resulting coronary heart
disease and cerebral stroke, is the most common cause of death
in industrialized nations. Mammalian cells have evolved
complex feedback mechanisms to ensure a sufficient supply of
cholesterol and to prevent its excessive accumulation.
During the process of atherosclerosis, these homeostatic
mechanisms fail in macrophages. Uncontrolled cholesterol
deposition is promoted by scavenger functions of the
macrophages and the adaptive mechanisms elicited are not sufficient
to process the lipid load. Consequently, a lipid-laden "foam
cell" is formed. The active stages of atherosclerotic lesions
are characterized by the extensive infiltration of
blood-derived monocyte/macrophages through the endothelium into
the arterial intima. The monocytes migrate into the arterial
intima where they internalize ox-LDL through scavenger
receptors and become macrophage-derived foam cells results
in the formation of fatty streaks, which are believed to
represent the earliest type of atherosclerotic plaque. CpG-ODN
can upregulate the related gene expression in foam cells,
which implies that CpG-ODN might function as a facilitative
atherosclerotic factor. This suggests that the clinical
application of CpG-ODN might be risky in cases of atherosclerosis.
Macrophage-derived foam cells in atherosclerotic lesions
are generally thought to play a major role in the pathology of
the disease. The uptake of ox-LDL by macrophages is a key
event implicated in the initiation and development of
atherosclerotic lesions. Two macrophage surface receptors, CD36
(a class B scavenger receptor) and the macrophage scavenger receptor (a class A scavenger receptor), have been
identified as the major receptors that bind and internalize
ox-LDL. CD36 is an 88 kDa transmembrane glycoprotein
that is expressed on monocytes, platelets, and
microvascular endothelium. The expression of CD36 in
monocytes/macrophages in tissue culture is dependent both on their
differentiation state and exposure to soluble mediators (cytokines
and growth factors). Modified lipoproteins such as LDL
immune complexes can also enter the macrophage
through the Fcg receptor, which is a classic inhibitory receptor that is
widely expressed on B cells, macrophages, neutrophils,
and mast cells. Once LDL immune complexes combine with the
Fcg receptor, it may enhance LDL oxidation and expression
of the scavenger receptor[21]. Lipoprotein lipase (LPL) is the
enzyme primarily responsible for the hydrolysis of core
triglycerides in circulating chylomicrons and very low density
lipoproteins (VLDL) to generate cholesterol-enriched
remnants[22]. LPL is synthesized by many cell types, including
vascular smooth muscle cells (VSMC) and macrophages, and
associates with the vascular endothelium. Increased LPL
activity in the arterial wall correlates with increased areas of
lipid deposition and increased atherosclerotic lesion
formation[23]. The activity of LPL in promoting atherosclerosis
may depend on its dual role as an enzyme and as a bridging
protein that increases the binding of lipoproteins to the cell
surface[22].
Our data suggest that CpG-ODN upregulates the
expression of CD36, Fcg2b, and LPL in foam cells, which indicates
that CpG-ODN might have some influence on the formation
of foam cells and related gene expression.
References
1 McCluskie MJ, Davis HL. CpG DNA is a potent enhancer of
systemic and mucosal immune responses against hepatitis B
surface antigen with intranasal administration to mice.
J Immunol 1998; 161: 4463-6.
2 Sommer F, Wilken H, Faller G, Lohoff M. Systemic Th1
immunization of mice against Helicobacter
pylori infection with CpG oligodeoxynucleotides as adjuvants does not protect from
infection but enhances gastritis. Infect Immun 2004; 72:
1029-35.
3 Weigel BJ, Rodeberg DA, Krieg AM, Blazar BR. CpG oligo-
deoxynucleotides potentiate the antitumor effects of
chemotherapy or tumor resection in an orthotopic murine model of
rhabdomyosarcoma. Clin Cancer Res 2003; 9: 3105-14.
4 Jorgensen JB, Johansen LH, Steiro K, Johansen A. CpG DNA
induces protective antiviral immune responses in Atlantic salmon
(Salmo salar L). J Virol 2003; 77: 11 471-9.
5 Sur S, Wild JS, Choudhury BK, Sur N, Alam R, Klinman DM.
Long term prevention of allergic lung inflammation in a mouse
model of asthma by CpG oligodeoxynucleotides. J Immunol
1999; 162: 6284-93.
6 Yi AK, Klinman DM, Martin TL, Matson S, Krieg AM. Rapid
immune activation by CpG motifs in bacterial DNA. Systemic
induction of IL-6 transcription through an antioxidant-sensitive
pathway. J Immunol 1996; 157: 5394-402.
7 Ballas ZK, Rasmussen WL, Krieg AM. Induction of NK activity
in murine and human cells by CpG motifs in oligodeoxynucleotides
and bacterial DNA. J Immunol 1996; 157: 1840-5.
8 Verthelyi D, Ishii KJ, Gursel M, Takeshita F, Klinman DM.
Human peripheral blood cells differentially recognize and respond
to two distinct CpG motifs. J Immunol 2001; 166: 2372-7.
9 Sparwasser T, Koch ES, Vabulas RM, Heeg K, Lipford GB, Ellwart
JW, et al. Bacterial DNA and immunostimulatory CpG
oligonucleotides trigger maturation and activation of murine dendritic
cells. Eur J Immunol 1998; 28: 2045-54.
10 Hu ZL, Sun SH, Zhou FJ. The binding of CpG-oligodeo-
xynucleotides to cell-surface and its immunostimulatory activity
are modulated by extracellular acidic pH. Vaccine 2003; 21:
485-90.
11 Vainio S, Ikonen E. Macrophage cholesterol transport: a critical
player in foam cell formation. Ann Med 2003; 35: 146-55.
12 Schena M, Salon D, Heller R. Parallel human genome analysis:
microarray-based expression monitoring of 1000 genes. Proc
Natl Acad Sci USA 1996; 93: 10 614-9.
13 Shalon D, Smith SJ, Brown PO. A DNA microarray system for
analyzing complex DNA samples using two-color fluorescent
probe hybridization. Genome Res 1996; 6: 639-45.
14 Kuzuya M, Naito M, Funaki C, Hayashi T, Asai K, Kuzuya F.
Lipid peroxide and transition metals are required for the toxicity
of oxidized low density lipoprotein to cultured endothelial cells.
Biochim Biophys Acta 1991; 1096: 155-61.
15 Yang PY, Rui YC, Li K, Huang XH, Jiang JM, Yu L. Expression
of intercellular adhesion molecule-1 in U937 foam cells and
inhibitory effect of imperatorin. Acta Pharmacol Sin 2002; 23:
327-30.
16 Hara A, Radin NS. Lipid extraction of tissues with a low-toxicity
solvent. Anal Biochem 1978; 90: 420-6.
17 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein
measurement with the Folin phenol reagent. J Biol Chem 1951; 193:
265-75.
18 Wang Z, Liu YL, Jiang ZS. A new method to measure the level of
L-arginine of aortic wall in rabbits. Chin J Arterioscler 1996; 4:
72-4.
19 Klinman DM, Yi AK, Beaucage SL, Conover J, Krieg AM. CpG
motifs present in bacteria DNA rapidly induce lymphocytes to
secrete interleukin 6, interleukin 12, and interferon gamma. Proc
Natl Acad Sci USA 1996; 93: 2879-83.
20 Horner AA, Raz E. Immunostimulatory sequence oligodeox-
ynucleotide-based vaccination and immunomodulation: two unique
but complementary strategies for the treatment of allergic
diseases. J Allergy Clin Immunol 2002; 110: 706-12.
21 Kiener PA, Rankin BM, Davis PM, Yocum SA, Warr GA, Grove
RI. Immune complexes of LDL induce atherogenic responses in
human monocytic cells. Arterioscler Thromb Vasc Biol 1995;
15: 990-9.
22 Goldberg IJ. Lipoprotein lipase and lipolysis: central roles in
lipoprotein metabolism and atherogenesis. J Lipid Res 1996; 37:
693-707.
23 Stein Y, Stein O. Lipoprotein lipase and atherosclerosis.
Atherosclerosis 2003; 170: 1-9.
|
