J. Lipid Res. Neurobiology of Lipids (ISSN1683-5506)
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1194/jlr.M600181-JLR200 on July 13, 2006

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
M600181-JLR200v1
47/10/2259    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Groeneweg, M.
Right arrow Articles by de Winther, M. P. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Groeneweg, M.
Right arrow Articles by de Winther, M. P. J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Journal of Lipid Research, Vol. 47, 2259-2267, October 2006
Copyright © 2006 by American Society for Biochemistry and Molecular Biology

Lipopolysaccharide-induced gene expression in murine macrophages is enhanced by prior exposure to oxLDL

Mathijs Groeneweg*, Edwin Kanters{dagger}, Monique N. Vergouwe*, Hilde Duerink*, Georg Kraal{dagger}, Marten H. Hofker* and Menno P. J. de Winther1,*

* Department of Molecular Genetics, Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, The Netherlands
{dagger} Department of Molecular Cell Biology and Immunology, Vrije Universiteit Medical Center, Amsterdam, The Netherlands

Published, JLR Papers in Press, July 13, 2006.

1 To whom correspondence should be addressed. e-mail: dewinther{at}gen.unimaas.nl


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS
 RESULTS
 DISCUSSION
 REFERENCES
 
Uptake of modified lipoproteins by macrophages results in the formation of foam cells. We investigated how foam cell formation affects the inflammatory response of macrophages. Murine bone marrow-derived macrophages were treated with oxidized LDL (oxLDL) to induce foam cell formation. Subsequently, the foam cells were activated with lipopolysaccharide (LPS), and the expression of lipid metabolism and inflammatory genes was analyzed. Furthermore, gene expression profiles of foam cells were analyzed using a microarray. We found that prior exposure to oxLDL resulted in enhanced LPS-induced tumor necrosis factor (TNF) and interleukin-6 (IL-6) gene expression, whereas the expression of the anti-inflammatory cytokine IL-10 and interferon-ß was decreased in foam cells. Also, LPS-induced cytokine secretion of TNF, IL-6, and IL-12 was enhanced, whereas secretion of IL-10 was strongly reduced after oxLDL preincubation. Microarray experiments showed that the overall inflammatory response induced by LPS was enhanced by oxLDL loading of the macrophages. Moreover, oxLDL loading was shown to result in increased nuclear factor-{kappa}B activation. In conclusion, our experiments show that the inflammatory response to LPS is enhanced by loading of macrophages with oxLDL. These data demonstrate that foam cell formation may augment the inflammatory response of macrophages during atherogenesis, possibly in an IL-10-dependent manner.

Supplementary key words cytokines • inflammation • modified LDL • lipopolysaccharide • RAW 264.7

Abbreviations: BMM, bone marrow-derived macrophage; CE, cholesteryl ester; IL-6, interleukin-6; LPS, lipopolysaccharide; NF-{kappa}B, nuclear factor-{kappa}B; oxLDL, oxidized LDL; qPCR, quantitative PCR; TNF, tumor necrosis factor


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS
 RESULTS
 DISCUSSION
 REFERENCES
 
Macrophages play a key role in atherosclerotic lesion development (1). The onset of atherosclerosis involves attraction of monocytes to the vessel wall at local sites of endothelial dysfunction. The monocytes subsequently migrate through the endothelium and differentiate into macrophages. These macrophages take up modified LDL particles, leading to the formation of foam cells, the first hallmark of an atherosclerotic lesion. Further progression of atherosclerosis involves the attraction of additional monocytes, influx of T-cells, proliferation and migration of smooth muscle cells, and processes like cell death by apoptosis or necrosis. Atherogenesis is accompanied by the local production and secretion of inflammatory mediators, for which the macrophage is a major source.

The main characteristic of foam cell formation is the accumulation of cholesteryl esters (CEs) from LDL taken up by the macrophages. In vitro, several forms of modified LDL are being studied; including copper-oxidized LDL (oxLDL), acetylated LDL, and aggregated LDL. When administered to macrophages, these forms of modified LDL are taken up and mainly stored as free cholesterol and CE, resulting in the formation of foam cells (2, 3).

Incubation of macrophages with low concentrations of oxLDL protects cells from apoptosis and induces cell proliferation (4–6). In contrast, high concentrations and long incubation times of oxLDL will induce cell death (5). The effects of oxLDL on the inflammatory capacity of macrophages are less clear. It is generally assumed that during foam cell formation, macrophages loose their ability to elicit a strong inflammatory response upon stimulation; however, results have not been consistent in different publications (7). Effects of oxLDL are dependent on intensity of LDL oxidation (8–10) and the amount of oxLDL applied to the cells. Low concentrations are thought to enhance the inflammatory response of macrophages after activation, whereas higher concentrations may repress the inflammatory response (7).

In our study, we were interested in the effects of oxLDL loading on lipopolysaccharide (LPS)-induced gene expression and on the inflammatory response of murine macrophages. To follow the transition of macrophages to foam cells and the effect of modified LDL on the inflammatory properties of macrophages, we performed real-time PCR and microarray experiments on murine bone marrow-derived macrophages (BMMs). Our results indicate that oxLDL loading of macrophages caused changes in cytokine gene expression in BMMs. In particular, oxLDL preincubation mainly enhanced the effects of LPS activation coinciding with an enhanced activation of nuclear factor-{kappa}B (NF-{kappa}B) and a downregulation of interleukin-10 (IL-10) and interferon-ß (IFN-ß).


   MATERIALS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS
RESULTS
 DISCUSSION
 REFERENCES
 
LDL isolation and modification
Serum from fresh blood (0.5 l, obtained from ‘De Stichting Sanquin Bloedvoorziening’) was obtained for LDL isolation, and LDL was oxidized by CuSO4 as described previously (11). The LDL and oxLDL were stored for up to 2 weeks under N2 at 4°C. Endotoxin concentration of all samples was below 5 pg/ml, as tested in a Limulus Amabocyte Lysate assay (HyCult Biotechnology b.v., The Netherlands).

Cell culture and stimulation
BMMs were obtained from C57/Bl6 mice as described previously (12, 13). After differentiation, cells were cultured in R10 (RPMI 1640 medium, containing 10% heat-inactivated fetal calf serum, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, and 10 mM Hepes) supplemented with 15% L-929 cell-conditioned medium (14). RAW 264.7 cells were grown in RPMI 1640 medium containing 10% heat-inactivated fetal calf serum, penicillin (100 U/ml), streptomycin (100 µg/ml), L-glutamine (2 mM) and ß-mercaptoethanol (0.1%). Experiments were performed in this same medium with the same setup as described for the BMMs. Cells were stimulated using 10 ng/ml LPS for 3 h. Cells were incubated with 25 µg/ml lipoprotein for the indicated times.

Lipid extraction and high-performance TLC
The BMMs were lifted from the bacterial plastic 6-well plates, suspended in 200 µl PBS, and frozen at –20°C. The cells were thawed and repeatedly passed through a syringe to fully lyse the cells. Cell lysate containing 200 µg protein was added to a total volume of 800 µl MilliQ. Chloroform (500 µl) and internal standard (20 µg cholesterol acetate/ml chloroform) were added, and the mixture was centrifuged at 3,000 rpm. The chloroform fraction was isolated, dried at 50°C under N2, and stored at –20°C. High-performance TLC (HPTLC) plates (Alltech/Applied Science) were activated overnight at 130°C. The plates were prerun with a 2:1 methanol-chloroform mixture, dried at 130°C, prerun with 10% NaOH in methanol, and activated overnight at 130°C. Lipid samples were dissolved in 50 µl chloroform and slowly applied to the HPTLC plate. The run was performed with a hexane-diethylether-2-propanol mixture (90:10:3). After the run, the plates were dried at 130°C and incubated in staining reagent (H2SO4-dissolved MnCL2 in 50% methanol) for 20 s, and incubated at 130°C for 30 min.

RNA isolation and quantitative PCR analysis
Total RNA was isolated from BMM using Tri-Reagent from Sigma according to the manufacturer's recommendation. RNA concentration was measured using the Biorad Smartspec 3000 photospectrometer. Total RNA (1 µg) was used to obtain cDNA. Oligo-dT primers and RevertAid M-MuLV reverse transcriptase (Fermentas) were used according to the manufacturer's recommendation. cDNA (10 ng) was used for TaqMan analysis with either the quantitative PCR (qPCR) Mastermix Plus Kit (Eurogentec) with 300 nM primer and 200 nM probe or the qPCR Mastermix Plus Kit for SYBR Green (Eurogentec) with 300 nM primer. Analysis was performed on an ABI Prism 7700 sequence detector. The housekeeping genes cyclophillin A and ß-actin were used to correct for RNA concentration differences between the samples. Primer sequences can be obtained upon request.

ELISA
ELISAs were performed according to the manufacturer's protocol. ELISA plates were obtained from NUNC (Denmark). Antibodies were detected by incubation with Ortho Phynyl Diamine (OPD)/H2O2, and H2SO4 was added to stop the reaction. Absorbance was measured at 490 nm in a BENCHMARK microplate reader (BIORAD).

Microarrays
Cy 3- or Cy 5-labeled cDNA (1 µg) was hybridized to slides containing National Institute on Aging mouse 15K v3 cloneset. Slides were obtained from The University Health Network in Toronto, Canada (15–17) (http://www.microarrays.ca). A detailed description of the procedure can be found online in the supplemental material.

Luciferase activity
BMM from NF-{kappa}B reporter mice (18) (a generous gift from Drs. H. Carlsen and R. Blomhoff, University of Oslo, Norway) or RAW 264.7 cells stably transfected with the 3x-{kappa}B-luc plasmid (18) were incubated for 0, 1, 12, 24, and 48 h with 25 µg/ml oxLDL and subsequently activated with 10 ng/ml LPS. Cells were lyzed using cell culture lysis buffer (Promega) for 20 min, after which 10 µl cell lysate was added to 50 µl luciferin. Luciferase activity was measured using the Lumac Biocounter M1500 luminometer.

Statistics
All data are expressed as a mean ± SEM of triplicate measures. Statistical analysis was performed using the Student's t-test, one-way ANOVA or two-way ANOVA when applicable, using GraphPad PRISM software. Significance is depicted in the respective figures. P values smaller than 0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS
 RESULTS
DISCUSSION
 REFERENCES
 
Analysis of foam cell formation
BMMs were incubated with oxLDL for 1, 12, 24, and 48 h to induce different stages of foam cell formation, then the cells were activated by LPS for an additional 3 h. Because we did not observe significant differences in gene expression patterns between 24 and 48 h of macrophage exposure to oxLDL, the 48 h incubation time point was omitted in the following experiments (data not shown). Cholesterol accumulation in response to oxLDL loading was monitored by lipid analysis using HPTLC. Free cholesterol concentration remained at the same level (data not shown), whereas the amount of intracellular CEs strongly increased over a period of 24 h (Fig. 1A ), indicating accumulation and processing of cholesterol in the BMMs. Cell death during oxLDL and LPS treatment was quantified by sub-G1 analysis. During oxLDL loading, the percentage of dead cells did not increase, and LPS stimulation for 3 h also did not have an effect on cell death (data not shown). qPCR experiments were performed to assess the metabolic responses of the BMMs to oxLDL. ABCA1, CD36, and SR-A expression increased after oxLDL administration (Fig. 1B–D). LPS stimulation further increased SR-A expression.


Figure 1
View larger version (21K):
[in this window]
[in a new window]
  		Fig. 1. Analysis of bone marrow-derived macrophages (BMMs) loaded with oxidized LDL (oxLDL) and activated with lipopolysaccharide (LPS). BMMs were incubated with 25 µg/ml oxLDL for the indicated times. A: High-performance TLC was used to quantify cholesteryl ester (CE) accumulation. CE accumulation was significant as tested by one-way ANOVA. B–D: Quantitative PCR (qPCR) analysis of the indicated genes after oxLDL incubation and LPS activation. Gene expression levels were corrected for cyclophillin A expression. Open bars indicate BMMs incubated with oxLDL only; shaded bars indicate BMMs incubated with oxLDL and subsequent LPS activation. Change in expression was significant, as tested by two-way ANOVA (P < 0.05). Graphs are representative of four experiments. Error bars represent SEM.

 
Inflammatory response of the macrophages
To analyze inflammatory responses, several genes were analyzed by qPCR. Interestingly, the expression level of tumor necrosis factor (TNF) and IL-6 after LPS stimulation was increased 2–3-fold by preincubation with oxLDL (Fig. 2A , B). Conversely, the induction of the anti-inflammatory cytokine IL-10 was strongly repressed in the cells after preincubation with oxLDL (Fig. 2C). Similarly, IL-12 was also decreased but only after 24 h oxLDL incubation (Fig. 2D). MCP1 and I{kappa}B{alpha} expression was induced after LPS stimulation but was not strongly affected by preloading with oxLDL (data not shown). These data show that oxLDL has both repressive and stimulating effects on LPS-induced inflammatory genes.


Figure 2
View larger version (13K):
[in this window]
[in a new window]
  		Fig. 2. Inflammatory responses in BMMs. A–D: Q-PCR analysis of the inflammatory genes after oxLDL incubation and LPS activation. Gene expression levels were corrected for cyclophillin A expression. Open bars indicate BMMs incubated with oxLDL only; shaded bars indicate BMMs incubated with oxLDL and subsequent LPS activation. Graphs are representative of four experiments. E: Luciferase assay on BMMs containing a nuclear factor-{kappa}B (NF-{kappa}B)-luciferase construct. Cells were preincubated with oxLDL for up to 24 h before LPS activation. All changes in expression and induction of the NF-{kappa}B-luciferase construct were significant as tested by two-way ANOVA (P < 0.05). Error bars represent SEM.

 
Because one of the major transcription factors involved in inflammatory gene expression is NF-{kappa}B, we focused on NF-{kappa}B-dependent gene expression. To investigate the involvement of NF-{kappa}B in the modulation of LPS-induced gene expression by oxLDL, an NF-{kappa}B-dependent luciferase assay was used. NF-{kappa}B activation was measured using BMMs from NF-{kappa}B reporter mice (18). After 24 h of oxLDL loading, there was a significant increase in LPS-induced luciferase activity compared with LPS-activated cells without oxLDL preloading (Fig. 2E). OxLDL incubation without LPS activation did not induce luciferase activity (Fig. 2E).

To examine whether the enhancement of the LPS-induced gene expression by oxLDL was specific for oxLDL, we also tested the expression of these genes in cells treated with native LDL. However, LPS-induced gene expression of several genes tested was not modulated by native LDL (data not shown).

Cytokine secretion in macrophages
To confirm the results obtained from qPCR experiments, we measured cytokine secretion to the medium after LPS activation. BMMs were treated for 1.5, 3, 6, and 24 h with 10 ng/ml LPS after preincubation with 25 µg/ml oxLDL for 24 h or without preincubation. The concentrations of TNF, IL-6, IL-12, and IL-10 in the medium were measured using an ELISA. In line with the qPCR experiments, TNF and IL-6 secretion upon LPS treatment was enhanced by oxLDL preincubation (Fig. 3A , B), whereas IL-10 secretion was reduced (Fig. 3C). Interestingly, whereas the expression of IL-12 was reduced in LPS-activated BMMs preincubated with oxLDL compared with LPS-activated BMMs (Fig. 2D) that were not preincubated with oxLDL, IL-12 secretion was enhanced by oxLDL preincubation (Fig. 3D). Other times of oxLDL loading (i.e., 3 h and 48 h) gave similar results with respect to an enhancement of TNF and IL-6 induction and a repression of IL-10 induction by LPS (data not shown).


Figure 3
View larger version (18K):
[in this window]
[in a new window]
  		Fig. 3. Cytokine secretion in activated macrophages. A–D: ELISA analysis of the inflammatory genes after oxLDL incubation and LPS activation. Open bars indicate BMMs activated with LPS only; shaded bars indicate BMMs incubated for 24 h with oxLDL and subsequent LPS activation. Numbers on the x-axes indicate duration of LPS activation in hours. Graphs are representative of three experiments. All changes in expression were significant as tested by two-way ANOVA (P < 0.05). Error bars represent SEM.

 
Microarray analysis
To examine the general effect of oxLDL preincubation on LPS-induced gene expression in an unbiased way, the macrophages were further investigated via microarray analysis. We used a design proposed by Yang and Speed (19) and Churchill (20), in which every oxLDL loading time point was compared with the previous, resulting in data showing a relative change of expression in comparison to the previous condition, giving a cumulative expression pattern for each gene (see supplementary Fig. I). In our data analysis, we used genes that were at least 1.5-fold up- or downregulated in at least one lipid-loaded condition. In total, the number of genes that met these criteria was 377. Many genes differentially regulated by LPS were influenced by oxLDL loading. Interestingly, genes upregulated by LPS mainly became further upregulated by oxLDL preincubation, whereas genes downregulated by LPS mainly became further downregulated by oxLDL preincubation (Table 1 ). This indicates that oxLDL mainly enhances the gene expression changes induced by LPS stimulation. To analyze the function of the genes found, genes were annotated using Gene Ontology (21) and the description at the National Center for Biotechnology Information's Entrez Gene (http://www.ncbi.nlm.nih.gov). Based on this annotation, we did not readily recognize genes known to be involved in lipid metabolism or inflammation. Please see http://www.jlr.org for genes found to be differentially expressed, their gene ontology, and the fold change in expression (also see supplemental Table I).


View this table:
[in this window]
[in a new window]
  		TABLE 1. Effects of oxLDL on LPS regulated genes

 
Effects of oxLDL loading on IL-10 target genes
Previous studies have shown that oxLDL loading highly affects the production of IFN-ß through inhibition of IFN-ß transcription (22). In addition, the induction of IFN-ß by LPS has been shown to be dependent on autocrine effects of secreted IL-10 (23). We could indeed show that oxLDL loading of BMMs reduced the induction of IFN-ß gene expression by LPS by approximately 50% (Fig. 4A ). Not only was IFN-ß expression repressed by oxLDL preincubation, IFN-ß secretion was also reduced. BMMs were preincubated with oxLDL for 24 h and subsequently activated with LPS for the indicated times. IFN-ß secretion was inhibited by oxLDL compared with cells that were not preincubated, independent of LPS activation time (Fig. 4B).


Figure 4
View larger version (17K):
[in this window]
[in a new window]
  		Fig. 4. Interleukin-10 (IL-10) target genes are downregulated by oxLDL. A: BMMs were incubated for 24 h with or without oxLDL and subsequently activated with LPS, and interferon-ß (IFN-ß) expression was quantified. B: BMMs were incubated for 24 h with oxLDL and subsequently activated by LPS for the indicated times (hours). OxLDL loading caused a decrease in IFN-ß secretion, independent of the duration of LPS activation, as tested by two-way ANOVA (P < 0.05). C–D: BMMs were incubated for 24 h with or without oxLDL and subsequently activated with LPS, and the expression of the IL-10 target genes IL-1RA and NOS2 was quantified. Gene expression was corrected for cyclophillin expression. A, C, and D: *P < 0.05. Error bars represent SEM.

 
To further investigate the effects of oxLDL on other IL-10-dependent genes, we analyzed additional genes that are known to be dependent on autocrine IL-10 action: IL-1 receptor antagonist (IL-1RA) and inducible NO synthase (NOS2 or iNOS) (23). Both were strongly induced by LPS (Fig. 4C, D), and this induction was indeed repressed by 24 h oxLDL preloading. Furthermore, we analyzed the IL-10-dependent genes Arginase 1 and Arginase 2. However, Arginase 1 was not induced by 3 h of LPS but was repressed by oxLDL loading. Arginase 2 showed an inhibition similar to that of IFN-ß, IL-1RA, and iNOS, but this effect did not reach statistical significance (data not shown). Taken together, these data indicate that the reduced LPS induction of IL-10 in oxLDL-treated macrophages may have autocrine effects on genes that are dependent on IL-10 secretion by the macrophage.

Effects of oxLDL on LPS-treated RAW 264.7 macrophages
To confirm the results we obtained in BMMs and to extend our studies to an alternative source of macrophages, experiments were performed in the murine macrophage cell line RAW 264.7. These cells were incubated with or without oxLDL for 24 h and then activated with LPS. CD36 was increased by oxLDL loading (Fig. 5A ), as a hallmark of foam cell formation. Because the major effects in BMMs were observed on TNF and IL-10, we analyzed these genes in the RAW cells. TNF induction was increased approximately 2-fold in cells that had been loaded with oxLDL for 24 h (Fig. 5B), whereas IL-10 expression was strongly repressed by the oxLDL loading (Fig. 5C). These data confirm our findings in BMMs and show that the inflammation-modulating effects observed for BMMs also hold true in the RAW 264.7 cell line. Finally, we could show that an NF-{kappa}B reporter stably transfected in RAW cells was also more induced by LPS after oxLDL loading, also confirming our findings in BMMs (Fig. 5D).


Figure 5
View larger version (19K):
[in this window]
[in a new window]
  		Fig. 5. OxLDL-induced gene expression in RAW 264.7 macrophages. Cells were incubated with 25 µg/ml oxLDL for 24 h and afterwards stimulated with LPS. Left sides of the graphs indicate cells that were not stimulated with LPS; right sides of the graphs indicate cells that were stimulated with LPS. A–C: Q-PCR analysis of genes induced by oxLDL and LPS. D: An NF-{kappa}B-dependent luciferase assay was used to investigate the effects of oxLDL on LPS-induced NF-{kappa}B activation; *P < 0.05. Error bars represent SEM.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we examined the effect of oxLDL loading on the inflammatory capacity of macrophages. We show that LPS-induced TNF and IL-6 expression and secretion were upregulated, whereas IL-10 expression and secretion were downregulated by oxLDL preincubation. Moreover, using microarray analysis, we found that the overall LPS-induced gene expression patterns were mainly enhanced by oxLDL preloading. These results indicate a shift toward a proinflammatory phenotype that may highly affect atherosclerotic lesion development (1, 24).

Previous studies have assessed the effects of modified lipids on macrophage inflammatory responses. In contrast to our finding that LPS-induced gene expression patterns were enhanced by prior exposure to oxLDL, a study done by Ohlssen et al. (25) showed that oxLDL loading reduced NF-{kappa}B activation and subsequent TNF and IL-1ß induction in human primary macrophages. Mikita et al. (26) recently performed a large-scale microarray analysis experiment on the human monocyte/macrophage cell line THP-1. They showed that 72 h of oxLDL loading strongly affects and mainly attenuates the LPS-induced inflammatory gene expression of macrophages and also inhibits NF-{kappa}B DNA binding. Interestingly, several major NF-{kappa}B-related proinflammatory genes, such as TNF, IL-1ß, and IL-8, were strongly upregulated in these experiments (26), similar to the regulation of TNF and IL-6 in our experiment. The enhancement of LPS-induced gene expression patterns has also previously been shown by others. Brand et al. (27) have described oxLDL enhancement of LPS-induced TF expression in human monocytes. It should be noted however, that they did not find this for TNF expression, indicating diverse effects on different signaling pathways.

A possible mechanism through which oxLDL could influence the inflammatory response is apoptosis. OxLDL may affect the macrophages either by inducing apoptosis as a result of the oxidation level of the lipoprotein (4, 9) or by inhibiting the uptake of apoptotic cells by macrophages, thereby prolonging the inflammatory response, as has been described by Kahn et al. (28). However, we did not see major changes in apoptosis in our experiments, and therefore the contribution of apoptotic macrophages to our observed phenotype is probably very limited.

To control the inflammatory response, inflammatory activation pathways are strongly regulated by autocrine feedback mechanisms. When activated, macrophages induce both pro- and anti-inflammatory cytokines that can affect other cells as well as the macrophages themselves. A major anti-inflammatory cytokine produced by macrophages is IL-10. IL-10 has been shown to inhibit activation of macrophages through autocrine mechanisms (29, 30). Moreover, IL-10 has been shown to be important in atherosclerosis; an IL-10 knockout mouse model shows severely increased atherosclerosis (31–33). We now show that oxLDL loading of macrophages strongly represses the induction of IL-10, and this may have major implications for the inflammatory properties of macrophages during atherogenesis. The inhibitory effect of oxLDL on LPS-induced IL-10 expression can also be observed in the downregulation of the IL-10-dependent, LPS-induced expression of IFN-ß, IL-1RA, and iNOS2 (23). This indicates that oxLDL loading indeed disrupts autocrine IL-10 signaling. The inhibition of the IL-10 pathway by oxLDL loading may lead to reduced autocrine deactivation of macrophages, giving the proinflammatory phenotype we observe in our experiments (30).

IFN-ß expression, like that of the genes mentioned earlier, is also IL-10 dependent (23). Activation of macrophages by LPS resulted in a strong induction of IFN-ß expression. This induction was reduced after oxLDL loading of murine macrophages, which is in line with the downregulation of the other IL-10-dependent genes as well as with previous findings in THP-1 cells showing reduced levels of IFN-ß expression and secretion upon oxLDL loading of THP-1 macrophages (22). Earlier studies have shown an important role for IFN-ß and the IFN-ß receptor (i.e., IFN-{alpha}/ß R) in autocrine regulation of macrophage responses (34, 35). Thus, IFN-ß would be an interesting target for further research in foam cell formation and atherosclerotic plaque development.

There may be many different forms of modified LDL present in atherosclerotic lesions. Our Cu2+-oxidized LDL is an often-used model substance for physiological modified LDL. However, it contains many different modifications of the lipid moiety and of the apolipoprotein B protein component of the LDL particle. Certain modified phospholipids, such as oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (oxPAPC), have been described as modulating the inflammatory response. Not only is oxPAPC found in atherosclerotic lesions (36, 37), modified phospholipids have also been shown to be capable of affecting endothelial cell activation (38, 39). Future investigations may focus on the question of which components of oxLDL exert the inflammation-enhancing effect we observe.

Understanding the mechanisms underlying foam cell formation and the effects on inflammation and the early onset of atherosclerosis remains important for understanding the development of atherosclerotic plaques. Our results show an increased inflammatory response to stimuli during the oxLDL-induced transition from macrophage to foam cell, which could contribute to enhanced inflammation in the vessel wall. Gaining more understanding of the molecular mechanisms behind the onset and progression of atherosclerosis both in vivo and in vitro is essential for the development of novel drugs for the prevention and treatment of atherosclerosis.


   ACKNOWLEDGMENTS
 
This work was supported by the Dutch Organization for Scientific Research [Grants NWO 902-26-194 (M.H.), NWO 906-02-075 (M.P.J.d.W.), and VIDI 917.66.329 (M.P.J.d.W.)] and the European Union [European Vascular Genomics Network Grant LSHM-CT-2003-503254 (M.H. and M.P.J.d.W.)].

Manuscript received April 21, 2006 and in revised form June 27, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS
 RESULTS
 DISCUSSION
 REFERENCES
 
  1. Libby, P. 2002. Inflammation in atherosclerosis. Nature. 420: 868–874.[CrossRef][Medline]

  2. Heinecke, J. W., A. G. Suits, M. Aviram, and A. Chait. 1991. Phagocytosis of lipase-aggregated low density lipoprotein promotes macrophage foam cell formation. Sequential morphological and biochemical events. Arterioscler. Thromb. 11: 1643–1651.[Abstract/Free Full Text]

  3. Brown, A. J., E. L. Mander, I. C. Gelissen, L. Kritharides, R. T. Dean, and W. Jessup. 2000. Cholesterol and oxysterol metabolism and subcellular distribution in macrophage foam cells. Accumulation of oxidized esters in lysosomes. J. Lipid Res. 41: 226–237.[Abstract/Free Full Text]

  4. Bjorkerud, B., and S. Bjorkerud. 1996. Contrary effects of lightly and strongly oxidized LDL with potent promotion of growth versus apoptosis on arterial smooth muscle cells, macrophages, and fibroblasts. Arterioscler. Thromb. Vasc. Biol. 16: 416–424.[Abstract/Free Full Text]

  5. Hundal, R. S., A. Gomez-Munoz, J. Y. Kong, B. S. Salh, A. Marotta, V. Duronio, and U. P. Steinbrecher. 2003. Oxidized low density lipoprotein inhibits macrophage apoptosis by blocking ceramide generation, thereby maintaining protein kinase B activation and Bcl-XL levels. J. Biol. Chem. 278: 24399–24408.[Abstract/Free Full Text]

  6. Sakai, M., S. Kobori, A. Miyazaki, and S. Horiuchi. 2000. Macrophage proliferation in atherosclerosis. Curr. Opin. Lipidol. 11: 503–509.[CrossRef][Medline]

  7. Robbesyn, F., R. Salvayre, and A. Negre-Salvayre. 2004. Dual role of oxidized LDL on the NF-kappaB signaling pathway. Free Radic. Res. 38: 541–551.[CrossRef][Medline]

  8. Carpenter, K. L., I. R. Challis, and M. J. Arends. 2003. Mildly oxidised LDL induces more macrophage death than moderately oxidised LDL: roles of peroxidation, lipoprotein-associated phospholipase A2 and PPARgamma. FEBS Lett. 553: 145–150.[CrossRef][Medline]

  9. Han, C. Y., and Y. K. Pak. 1999. Oxidation-dependent effects of oxidized LDL: proliferation or cell death. Exp. Mol. Med. 31: 165–173.[Medline]

  10. Kavanagh, I. C., C. E. Symes, P. Renaudin, E. Nova, M. D. Mesa, G. Boukouvalas, D. S. Leake, and P. Yaqoob. 2003. Degree of oxidation of low density lipoprotein affects expression of CD36 and PPARgamma, but not cytokine production, by human monocyte-macrophages. Atherosclerosis. 168: 271–282.[CrossRef][Medline]

  11. Hendriks, W. L., H. van der Boom, L. C. van Vark, and L. M. Havekes. 1996. Lipoprotein lipase stimulates the binding and uptake of moderately oxidized low-density lipoprotein by J774 macrophages. Biochem. J. 314: 563–568.[Medline]

  12. Peiser, L., P. J. Gough, T. Kodama, and S. Gordon. 2000. Macrophage class A scavenger receptor-mediated phagocytosis of Escherichia coli: role of cell heterogeneity, microbial strain, and culture conditions in vitro. Infect. Immun. 68: 1953–1963.[Abstract/Free Full Text]

  13. Kanters, E., M. Pasparakis, M. J. Gijbels, M. N. Vergouwe, I. Partouns-Hendriks, R. J. Fijneman, B. E. Clausen, I. Forster, M. M. Kockx, K. Rajewsky, et al. 2003. Inhibition of NF-kappaB activation in macrophages increases atherosclerosis in LDL receptor-deficient mice. J. Clin. Invest. 112: 1176–1185.[CrossRef][Medline]

  14. Hume, D. A., and S. Gordon. 1983. Optimal conditions for proliferation of bone marrow-derived mouse macrophages in culture: the roles of CSF-1, serum, Ca2+, and adherence. J. Cell. Physiol. 117: 189–194.[CrossRef][Medline]

  15. Tanaka, T. S., T. Kunath, W. L. Kimber, S. A. Jaradat, C. A. Stagg, M. Usuda, T. Yokota, H. Niwa, J. Rossant, and M. S. Ko. 2002. Gene expression profiling of embryo-derived stem cells reveals candidate genes associated with pluripotency and lineage specificity. Genome Res. 12: 1921–1928.[Abstract/Free Full Text]

  16. Cui, C. Y., M. Durmowicz, T. S. Tanaka, A. J. Hartung, T. Tezuka, K. Hashimoto, M. S. Ko, A. K. Srivastava, and D. Schlessinger. 2002. EDA targets revealed by skin gene expression profiles of wild-type, Tabby and Tabby EDA-A1 transgenic mice. Hum. Mol. Genet. 11: 1763–1773.[Abstract/Free Full Text]

  17. Tanaka, T. S., S. A. Jaradat, M. K. Lim, G. J. Kargul, X. Wang, M. J. Grahovac, S. Pantano, Y. Sano, Y. Piao, R. Nagaraja, et al. 2000. Genome-wide expression profiling of mid-gestation placenta and embryo using a 15,000 mouse developmental cDNA microarray. Proc. Natl. Acad. Sci. USA. 97: 9127–9132.[Abstract/Free Full Text]

  18. Carlsen, H., J. O. Moskaug, S. H. Fromm, and R. Blomhoff. 2002. In ivo imaging of NF-{kappa}B activity. J. Immunol. 168: 1441–1446.[Abstract/Free Full Text]

  19. Yang, Y. H., and T. Speed. 2002. Design issues for cDNA microarray experiments. Nat. Rev. Genet. 3: 579–588.[CrossRef][Medline]

  20. Churchill, G. A. 2002. Fundamentals of experimental design for cDNA microarrays. Nat. Genet. 32 (Suppl.): 490–495.[CrossRef][Medline]

  21. Ashburner, M., C. A. Ball, J. A. Blake, D. Botstein, H. Butler, J. M. Cherry, A. P. Davis, K. Dolinski, S. S. Dwight, J. T. Eppig, et al. 2000. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat. Genet. 25: 25–29.[CrossRef][Medline]

  22. Marson, A., R. M. Lawn, and T. Mikita. 2004. Oxidized low density lipoprotein blocks lipopolysaccharide-induced interferon beta synthesis in human macrophages by interfering with IRF3 activation. J. Biol. Chem. 279: 28781–28788.[Abstract/Free Full Text]

  23. Lang, R., D. Patel, J. J. Morris, R. L. Rutschman, and P. J. Murray. 2002. Shaping gene expression in activated and resting primary macrophages by IL-10. J. Immunol. 169: 2253–2263.[Abstract/Free Full Text]

  24. Hansson, G. K. 2005. Inflammation, atherosclerosis, and coronary artery disease. N. Engl. J. Med. 352: 1685–1695.[Free Full Text]

  25. Ohlsson, B. G., M. C. Englund, A. L. Karlsson, E. Knutsen, C. Erixon, H. Skribeck, Y. Liu, G. Bondjers, and O. Wiklund. 1996. Oxidized low density lipoprotein inhibits lipopolysaccharide-induced binding of nuclear factor-kappaB to DNA and the subsequent expression of tumor necrosis factor-alpha and interleukin-1beta in macrophages. J. Clin. Invest. 98: 78–89.[Medline]

  26. Mikita, T., G. Porter, R. M. Lawn, and D. Shiffman. 2001. Oxidized low density lipoprotein exposure alters the transcriptional response of macrophages to inflammatory stimulus. J. Biol. Chem. 276: 45729–45739.[Abstract/Free Full Text]

  27. Brand, K., C. L. Banka, N. Mackman, R. A. Terkeltaub, S. T. Fan, and L. K. Curtiss. 1994. Oxidized LDL enhances lipopolysaccharide-induced tissue factor expression in human adherent monocytes. Arterioscler. Thromb. 14: 790–797.[Abstract/Free Full Text]

  28. Khan, M., S. Pelengaris, M. Cooper, C. Smith, G. Evan, and J. Betteridge. 2003. Oxidised lipoproteins may promote inflammation through the selective delay of engulfment but not binding of apoptotic cells by macrophages. Atherosclerosis. 171: 21–29.[CrossRef][Medline]

  29. O'Farrell, A. M., Y. Liu, K. W. Moore, and A. L. Mui. 1998. IL-10 inhibits macrophage activation and proliferation by distinct signaling mechanisms: evidence for Stat3-dependent and -independent pathways. EMBO J. 17: 1006–1018.[CrossRef][Medline]

  30. Lang, R., R. L. Rutschman, D. R. Greaves, and P. J. Murray. 2002. Autocrine deactivation of macrophages in transgenic mice constitutively overexpressing IL-10 under control of the human CD68 promoter. J. Immunol. 168: 3402–3411.[Abstract/Free Full Text]

  31. Pinderski Oslund, L. J., C. C. Hedrick, T. Olvera, A. Hagenbaugh, M. Territo, J. A. Berliner, and A. I. Fyfe. 1999. Interleukin-10 blocks atherosclerotic events in vitro and in vivo. Arterioscler. Thromb. Vasc. Biol. 19: 2847–2853.[Abstract/Free Full Text]

  32. Mallat, Z., S. Besnard, M. Duriez, V. Deleuze, F. Emmanuel, M. F. Bureau, F. Soubrier, B. Esposito, H. Duez, C. Fievet, et al. 1999. Protective role of interleukin-10 in atherosclerosis. Circ. Res. 85: e17–e24.[Abstract/Free Full Text]

  33. Caligiuri, G., M. Rudling, V. Ollivier, M. P. Jacob, J. B. Michel, G. K. Hansson, and A. Nicoletti. 2003. Interleukin-10 deficiency increases atherosclerosis, thrombosis, and low-density lipoproteins in apolipoprotein E knockout mice. Mol. Med. 9: 10–17.[Medline]

  34. Hwang, S. Y., P. J. Hertzog, K. A. Holland, S. H. Sumarsono, M. J. Tymms, J. A. Hamilton, G. Whitty, I. Bertoncello, and I. Kola. 1995. A null mutation in the gene encoding a type I interferon receptor component eliminates antiproliferative and antiviral responses to interferons alpha and beta and alters macrophage responses. Proc. Natl. Acad. Sci. USA. 92: 11284–11288.[Abstract/Free Full Text]

  35. Jacobs, A. T., and L. J. Ignarro. 2001. Lipopolysaccharide-induced expression of interferon-beta mediates the timing of inducible nitric-oxide synthase induction in RAW 264.7 macrophages. J. Biol. Chem. 276: 47950–47957.[Abstract/Free Full Text]

  36. Leitinger, N. 2003. Oxidized phospholipids as modulators of inflammation in atherosclerosis. Curr. Opin. Lipidol. 14: 421–430.[CrossRef][Medline]

  37. Berliner, J. A., G. Subbanagounder, N. Leitinger, A. D. Watson, and D. Vora. 2001. Evidence for a role of phospholipid oxidation products in atherogenesis. Trends Cardiovasc. Med. 11: 142–147.[CrossRef][Medline]

  38. Bluml, S., S. Kirchberger, V. N. Bochkov, G. Kronke, K. Stuhlmeier, O. Majdic, G. J. Zlabinger, W. Knapp, B. R. Binder, J. Stockl, et al. 2005. Oxidized phospholipids negatively regulate dendritic cell maturation induced by TLRs and CD40. J. Immunol. 175: 501–508.[Abstract/Free Full Text]

  39. Bochkov, V. N., D. Mechtcheriakova, M. Lucerna, J. Huber, R. Malli, W. F. Graier, E. Hofer, B. R. Binder, and N. Leitinger. 2002. Oxidized phospholipids stimulate tissue factor expression in human endothelial cells via activation of ERK/EGR-1 and Ca(++)/NFAT. Blood. 99: 199–206.[Abstract/Free Full Text]


Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
D. M. J. Curfs, S. A. I. Ghesquiere, M. N. Vergouwe, I. van der Made, M. J. J. Gijbels, D. R. Greaves, J. S. Verbeek, M. H. Hofker, and M. P. J. de Winther
Macrophage Secretory Phospholipase A2 Group X Enhances Anti-inflammatory Responses, Promotes Lipid Accumulation, and Contributes to Aberrant Lung Pathology
J. Biol. Chem., August 1, 2008; 283(31): 21640 - 21648.
[Abstract] [Full Text] [PDF]

Home page
 J. Lipid Res.Home page
P. M. Hutchins, R. M. Barkley, and R. C. Murphy
Separation of cellular nonpolar neutral lipids by normal-phase chromatography and analysis by electrospray ionization mass spectrometry
J. Lipid Res., April 1, 2008; 49(4): 804 - 813.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
M600181-JLR200v1
47/10/2259    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Groeneweg, M.
Right arrow Articles by de Winther, M. P. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Groeneweg, M.
Right arrow Articles by de Winther, M. P. J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Journal of Biological Chemistry 
 Molecular and Cellular Proteomics   ASBMB Today