- Email: [email protected]
2,4-Decadienal downregulates TNF-α gene expression in THP-1 human macrophages
Atherosclerosis 158 (2001) 95 – 101 www.elsevier.com/locate/atherosclerosis
2,4-Decadienal downregulates TNF-a gene expression in THP-1 human macrophages Josefa Girona, Joan-Carles Vallve´, Josep Ribalta, Mercedes Heras, Sı´lvia Olive´, Lluı´s Masana * Unitat de Recerca de Lı´pids i Arteriosclerosi, Facultat de Medicina, Uni6ersitat Ro6ira i Virgili, C/Sant Llorenc¸ 21, 43201 Reus, Spain Received 6 July 2000; received in revised form 27 December 2000; accepted 3 January 2001
Abstract Oxidized lipoproteins inhibit TNF-a secretion by human THP-1 macrophages due, at least in part, to aldehydes derived from the oxidation of polyunsaturated fatty acids. This study extends these findings by investigating the effect of three aldehydes (2,4-decadienal (2,4-DDE), hexanal and 4-hydroxynonenal (4-HNE)) on TNF-a and IL-1b mRNA expression. The 2,4-DDE and 4-HNE showed considerable biological activity which induced cytotoxicity on THP-1 macrophages at concentration of 50 mM. Hexanal, on the other hand, had a lower cytotoxic capacity and concentration of 1000 mM was needed for the effect to be observed. Exposure of THP-1 macrophages to aldehydes for 24 h inhibited TNF-a mRNA expression but increased or did not affect IL-1b mRNA levels. The inhibitory action of 2,4-DDE was dose dependent and began at 5 mM (46%, P B 0.001). The effect of 4-HNE was less inhibitory than 4-DDE but only when cytotoxic concentrations were used (50 mM). Very high concentrations of hexanal (200 mM) were needed to inhibit TNF-a expression (23%, PB 0.001). This downregulation of TNF-a gene expression by 2,4-DDE was parallel to a lower protein production. These data indicate that low levels of 2,4-DDE may modulate inflammatory action by inhibiting TNF-a mRNA gene expression and that the biological activity of 2,4-DDE may be involved in the development of atherosclerosis. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Lipid peroxidation; Aldehydes; 2,4-Decadienal; THP-1 human macrophages; TNF-a; Gene expression; Atherosclerosis
1. Introduction There is increasing evidence to suggest that lipoprotein oxidation plays an important role in the pathogenesis of atherosclerosis [1]. Oxidation is one of the most likely ways of modifying low density lipoprotein (LDL) in vivo. However, oxidized LDL (ox-LDL) is a complex mixture of various components including lipid hydroperoxides, oxysterols and aldehydes [2]. Of these, some aldehydes diffuse from lipoproteins and induce biological effects by themselves. Core aldehydes are present in human atherosclerotic lesions [3]. Malondialdehyde (MDA), 4-HNE and hexanal are some of the aldehydes in ox-LDL and are chemotatic for human monocyte-macrophages [4]. 4-HNE induces vascular * Corresponding author. Tel.: + 34-977-759318; fax: +34-977759322. E-mail address: [email protected] (L. Masana).
smooth muscle cell growth by stimulating c-fos and c-jun expression [5] and can bind directly to membrane regulatory proteins [6]. The covalent binding of aldehydes with apolipoprotein B leads to the formation of immunogenic structures (4-HNE, MDA) that are recognized by the scavenger receptors of macrophages in ox-LDL [7]. This, in turn, leads to the formation of foam cells. Some aldehydes can also induce potent cytotoxic effects [8]. Oxidized lipoproteins can promote or suppress the expression of inflammatory cytokines in endothelial cells and mononuclear phagocytes [9–13] and it has been reported that suppression of TNF-a expression by ox-LDL is contained within the lipid fraction [14]. We have previously demonstrated that the fact that oxidized lipoproteins inhibit TNF-a secretion is partly due to aldehydes [15]. The effect of 2,4-DDE, which inhibits TNF-a and stimulates IL-1b secretion, is particularly strong. It also induces considerably more cytotoxicity than other aldehydes and hydroperoxides [16].
0021-9150/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 2 1 - 9 1 5 0 ( 0 1 ) 0 0 4 1 9 - 1
96
J. Girona et al. / Atherosclerosis 158 (2001) 95–101
Because of the role of these aldehydes might have in the pathogenesis of atherosclerosis, in this paper we discuss how 2,4-DDE and other aldehydes affect, on the mRNA level, the modulation of TNF-a and IL-1b in macrophages.
2. Materials and methods
2.1. Cell culture The human monocytic leukemia cell line THP-1 [17] was obtained from the American Type Culture Collection (ATCC c TIB 202) and maintained in RPMI1640 (Gibco-BRL) medium supplemented with heat-inactivated 10% FBS (Gibco-BRL, Myoclone Super Plus FBS, USA), 100 U/l penicillin and 100 mg/ml streptomycin. 2-mercaptoethanol was added to a final concentration of 0.5 mM and the cells were placed in a humidified incubator at 37°C and 5% CO2 until there were enough cells available for experiments. The cells were pelleted by centrifugation and plated at a final concentration of 1× 106 cells/ml in 12 multiwell dishes (Nalge Nunc Int., USA). Phorbol 12-myristate 13-acetate (PMA) at a final concentration of 50 ng/ml in dimethyl sulfoxide (DMSO) (final concentration 0.1%) was added to the medium to differentiate monocytes from macrophages [17]. During macrophage differentiation, TNF-a expression has a biphasic behaviour with a maximum at 2 h when cells begin to secrete protein (data not shown). After 72 h incubation with PMA, the cells were washed three times with PBS and fresh PMA free medium was added. Cells were used for the experiments after 2 days of incubation with complete medium and without PMA, in order to do the experiments in non-high activated cells. Basal levels of TNF-a in these cells were lower than after differentiation with PMA. Incubations were done in medium supplemented with heat-inactivated 10% FBS to avoid toxicity.
2.2. Cytotoxicity The amount of LDH released into the medium determined the cytotoxic effect and it was measured in a Cobas – Mira autoanalyzer (Roche, Switzerland) using an enzymatic method (Boehringer Mannheim, GmbH, Germany), optimized according to the recommendation of the Deutsche Gesellschaf for Klinische Chemie. This method is based on the direct, NADH2-coupled assay which uses pyruvate as the substrate. The absorbance was read at 340 nm. Results are expressed as LDH leakage (%), which represents the amount in the supernatant plus the cells as determined following cell lysis (1% triton). Cells were also visualized under phase-contrast microscopy so that morphological changes could be assessed.
2.3. Dose response experiments Macrophages were incubated with 2,4-DDE at concentrations of 0.05, 0.5, 1, 5, 10, 25 and 50 mM; hexanal at concentrations of 1, 25, 50, 100, 200, 500 and 1000 mM; and 4-HNE at concentrations of 0.5, 1, 5, 10, 20 and 50 mM for 24 h. A stock solution of each commercial pure aldehyde (Aldrich) was prepared. The final concentration of ethanol in the culture media was less than 0.5%. Cells incubated with vehicle alone are designated as untreated cells. After the incubations, the media were collected and centrifuged for 5 min at 900 rpm in order to discard non-adherent cells and cell debris. An aliquot of media was taken so that LDH could be measured immediately. Cells were used for total RNA extraction and IL-1b and TNF-a expression. For TNF-a protein studies macrophages were incubated with 2,4-DDE at concentrations of 0, 10, 20 and 50 mM for 24 h. Media were collected and stored at − 20°C and the TNF-a protein was determined within 1 month.
2.4. 2,4 DDE time response experiments Macrophages were incubated with 2,4-DDE (25 mM) and lipopolysaccharide (100 ng/ml) (E. coli LPS; serotype 026:B6, Sigma) for 0, 2, 6 and 24 h in triplicate. LPS was the positive control for cytokine expression. After the incubations, the media were discarded and the total RNA was extracted from the cells.
2.5. Analysis of TNF-h and IL-1i mRNA Total cellular RNA was isolated from the cells by the Ultraspec RNA isolation system (Biotecx Lab., Inc., USA). The oligonucleotide primers (Life Technologies, USA) for TNF-a were 5% CCT TGG TCT GGT AGG AGA CG 3% and 5% CAG AGG GAA GAG TTC CCC AG 3% and for IL-1b they were 5% TGG AGA ACA CCA CTT GTT GCT CCA 3% and 5% AAA CAG ATG AAG TGC TCC TTC CAG G 3%. These were the ones suggested by Wang et al. [18]. A semi-quantitative RT-PCR method was used to determine TNF-a and IL-1b mRNA using the SuperScript one-step RT-PCR system (Life Technologies, USA). Fifty nanogram of total RNA were used. cDNA synthesis and pre-denaturation were performed at 55°C for 30 min and then at 94°C for 2 min. For TNF-a, the PCR amplification consisted of first denaturing the DNA chains at 94°C for 30 s, then annealing them at 57°C for 30 s and finally extending them at 72°C for 30 s and 32 cycles. For IL-1b, the PCR amplification consisted of first denaturing the DNA chains at 94°C for 30 s, then annealing them at 55°C for 30 s and finally extending them at 72°C for 30 s and 36 cycles. There was a final
J. Girona et al. / Atherosclerosis 158 (2001) 95–101
extension at 72°C for 7 min. The b-actin gene was used as the loading control. All incubations were carried out in a Perkin-Elmer 2400 Thermal Cycler. The PCR products were separated on a 2% agarose gel. Ten microliter of each PCR mixture (25 ml) was used for electrophoresis which was run at 70 V for 45 min. The bands were visualized with ethidium bromide and the net intensity of each band was analyzed in the Kodak Digital Science 1D program. Results are expressed as the relative mRNA intensity and the mean cytokine/bactin net intensity ratio.
97
3.2. TNF-h and IL-1i mRNA expression studies Fig. 3 shows the effect of aldehydes on TNF-a and IL-1b mRNA levels. The dose-response experiments showed that either 2,4-DDE, 4-HNE or hexanal inhibited TNF-a mRNA expression after 24 h of incubation. In contrast, these aldehydes had no statistically significant effect on IL-1b mRNA levels at non-cytotoxic concentrations (Fig. 3). Among all the aldehydes tested, 2,4-DDE was the strongest inhibitor of TNF-a mRNA levels. Thus, addition of 2,4-DDE at concentration of 5
2.6. Determination of TNF-h and IL-1i protein secretion Cytokines were measured in the culture medium using commercial enzyme-linked immunosorbent assays (ELISA, Endogen, Inc., USA). The concentration of TNF-a was calculated as pg/ml using recombinant human TNF-a as standard at a concentration between 25.6 and 1000 pg/ml. The concentration of IL-1b was calculated as pg/ml using recombinant human IL-1b as standard at a concentration between 3.9 and 125 pg/ml. Blank values were obtained by analyzing a cell-free medium. Results are expressed as a percentage of those obtained with the blank.
2.7. Statistics The results are presented as means9 S.E.M. The statistical significance between two groups was determined by using the Student’s t-test. Various groups were compared by ANOVA and the statistical significance was calculated using the Bonferroni multiple comparison test. Statistical significance was set at a level of PB0.05. The Statistical Package for the Social Sciences (SPSS) software package version 8.0 was used for statistical analysis.
3. Results
3.1. Cytotoxicity e6aluation Fig. 1 shows the cytotoxic effects of 2,4-DDE, hexanal and 4-HNE on THP-1 macrophages. After 24 h of incubation with aldehydes, LDH release increased significantly with respect to the controls at a concentration of 50 mM for 2,4-DDE and 4-HNE and of 1000 mM for hexanal (P B 0.001), whereas lower concentrations of these aldehydes showed no cytotoxicity on macrophages. The cytotoxic effects were also confirmed at the same concentrations by the direct observation of morphological changes under phase-contrast microscopy (Fig. 2).
Fig. 1. Cytotoxicity of aldehydes. THP-1 macrophages were incubated with 0 – 50 mM of 2,4-DDE (A); 0 – 1000 mM of hexanal (B); 0 – 100 mM of 4-HNE (C), for 24 h. Results are expressed as LDH leakage (%) that represents the amount in the supernatant plus the cells as determined following cell lysis (1% triton). The data are shown as the mean9SEM of three experiments. *PB 0.001, compared to untreated cells.
98
J. Girona et al. / Atherosclerosis 158 (2001) 95–101
shows that non-cytotoxic concentrations did not modifiy IL-1b protein levels. We have also analysed by HPLC the content of 2,4-DDE in the medium after the incubation and we observed that 2,4-DDE remains in the medium when cells are incubated with 50 mM, suggesting that the effect observed was due to the direct action of free aldehyde (data not shown).
4. Discussion
Fig. 2. Phase-contrast microscopy showing the morphological changes of THP-1 macrophages after 24 h in absence (A) or presence (B) of 50 mM of 2,4-DDE, (X100).
mM significantly inhibited TNF-a mRNA levels by 46% (PB 0.001). This effect was greatest at 25 mM (88%, P B 0.001). 4-HNE had an inhibitory effect only when cytotoxic concentrations (50 mM) were used and the inhibition was lower than 2,4-DDE at the same concentration (32%, PB 0.001). Hexanal induced inhibition of TNF-a mRNA levels when very high concentrations were used in the experiments (]200 mM) and the percentage of inhibition was clearly lower (23%, PB 0.001) than that observed 2,4-DDE at 5 mM. Fig. 4 shows the results of the time-response experiments of 2,4-DDE. When compared to basal expression, 2,4-DDE at concentration of 25 mM induced a maximum inhibition effect on THP-1 macrophages at 2 h of incubation. A maximum inhibition was observed at 2 h (78%), with a recovery at 24 h (43%). This effect was completely different from that induced by LPS (100 ng/ml) used as a positive control in these experiments.
3.3. 2,4 -DDE suppresses the secretion of TNF-h Incubation of THP-1 macrophages with 2,4-DDE not only inhibited TNF-a mRNA levels but also reduced the secretion of the protein into the medium in a dose-dependent manner. This effect was observed at a concentration of 10 mM (33%) and reached statistical significance at 20 mM (68%, PB 0.05) (Fig. 5A). Fig. 5B
There is a great body of evidence to suggest that oxidized lipoproteins play an important role in the pathogenesis of atherosclerosis [1]. However the mechanisms involved in this process are not fully understood. Oxidized lipoproteins are taken up by macrophages that become foam cells. Minimally oxidized LDL also seems to participate in the endothelial activation and recruits inflammatory cells in the intima of the vessel wall [19]. Oxidized lipoproteins also participate in the initiation and propagation of the inflammatory process by modulating the expression of several genes involved in the arteriosclerotic process [20]. The oxidized lipoprotein is probably a mixture of particles with different degrees of oxidation. Furthermore, there are many different products derived from lipid peroxidation that have important biological actions as oxysterols derived from cholesterol oxidation, lysophosphatilcholine derived from phospholipid oxidation, hydroperoxides and aldehydes derived from fatty acid oxidation [21– 23]. These products can remain in the particle after the oxidative process but it is more likely that most of them will leave the lipoprotein and induce their biological effects away from the particle. It has also been demonstrated that the effect of oxidized lipoproteins on inflammation is variable and appears to depend, in part, on the extent of oxidation [16,24]. It is important to identify the molecules responsible for the biological effect of the oxidized lipoprotein. Previous reports have shown that extensively oxidized LDL and HDL can inhibit TNF-a secretion by macrophages [11,15]. This effect was mainly mediated by some aldehydes derived from the oxidation of polyunsaturated fatty acids, and 2,4-DDE in particular was seen to have a very powerful effect. The present study discusses whether 2,4-DDE acts at the genetic level and analyzes its effect on TNF-a and IL-1b expression in THP-1 human macrophages. At lower concentrations, the cytotoxic activity of 2,4-DDE was much stronger than hexanal and 4-HNE. At non-toxic concentrations, however, 2,4DDE clearly inhibited TNF-a mRNA expression, unlike the case with IL-1b. In the same system LPS increased the expression of the TNF-a gene. The inhibition by 2,4-DDE could be observed at concentrations of less than 1 mM although it reached statistical signifi-
J. Girona et al. / Atherosclerosis 158 (2001) 95–101
cance at 5 mM and its maximum effect was at 25 mM. Hexanal required cytotoxic concentrations that were 200 times higher than those for 2,4-DDE to induce the same level of inhibition. The 4-HNE also produced a small inhibitory effect at cytotoxic concentrations. The inhibition was maximum after 2 h of aldehyde incubation and after 24 h there was a partial recovery. Like the mRNA expression, the production of protein also decreased and at a concentration of 20 mM was finally reduced by 60%. Although there are no quantitative data on the concentration of aldehydes in the artery wall, their presence has been demonstrated by the presence of antibodies against these substances [25]. Our results strongly suggest that lipoprotein oxidation is something other than just lipoprotein modification. Many molecules derived from the lipid oxidation process are responsible for the effects independently of the lipoprotein modification. Exactly why in vitro oxidized lipoproteins inhibit TNF-a is not clear but probably depends on the degree of lipoprotein oxidation. It has
99
been observed that minimally oxidized LDL can increase the production of TNF-a [26], while extensively oxidized LDL suppresses it [11,15]. The mechanisms by which 2,4-DDE repress TNF-a gene expression are not known but it probably interacts with some transcription factors. A recently published report demonstrates that 4-HNE specifically inhibits the activation of NFkB [27]. It has also been shown that the interaction of oxysterols with NF-kB, and AP-1 inhibits the TNF-a gene [14]. An interesting hypothesis is that 2,4-DDE derived from fatty acid oxidation could interact with PPAR-g since PPAR-g agonists specifically inhibit TNF-a promoter-regulated gene expression [28]. It has also been demonstrated recently that products of PUFAs oxidation, 9-HODE and 13-HODE are endogenous activators and ligands of PPAR-g [29]. Our data show that the biological activity of 2,4-DDE may have some pathogenic implications in the development of atherosclerosis and therefore, it could be considered as a new therapeutical target.
Fig. 3. Dose-response of 2,4-DDE on the TNF-a and IL-1b gene expression by THP-1 macrophages. Cells were incubated with 0 – 50 mM of 2,4-DDE (A); 0 – 1000 mM of hexanal (B); and 0 –50 mM of 4-HNE (C), for 24 h. Total cellular RNA was isolated as described in the materials and methods section. Expression of TNF-a and IL-1b was evaluated by semiquantitative RT-PCR using the a-actin gene as control. Results are expressed as the percentage of the relative mRNA intensity with respect to untreated cells. Control values for 2,4-DDE, hexanal and 4-HNE were 1.239 0.08, 1.27 9 0.10 and 1.10 90.01 mRNA Intensity Ratio for TNF-a and 1.60 9 0.09, 1.47 9 0.01 and 1.33 90.05 mRNA Intensity Ratio for IL1-b, respectively. The data are shown as the mean 9 S.E.M of three experiments. *PB 0.05, **PB 0.001 compared to the untreated cells.
100
J. Girona et al. / Atherosclerosis 158 (2001) 95–101
Acknowledgements This work was supported by a grant from the Fondo de Investigacio´ n Sanitaria (FIS 98/0228).
References
Fig. 4. Time-course of 2,4-DDE and LPS on the TNF-a gene expression by THP-1 macrophages. Cells were incubated with 25 mM 2,4-DDE or 100 ng/ml LPS up to 24 h. Total cellular RNA was isolated as described in Section 2. Expression of TNF-a was evaluated by semiquantitative RT-PCR using the a-actin gene as control. Results are expressed as the percentage of the relative mRNA intensity with respect to the control for each time point. The data are shown as the mean 9S.E.M of two experiments.
Fig. 5. Effect of 2,4-DDE on cytokine secretion. Cells were incubated with 10, 20 and 50 mM of 2,4-DDE for 24 h. After incubation, media were collected and TNF-a (A) and IL-1b (B) was measured with an ELISA kit. Results are expressed as pg/ml. The data are shown as the mean 9 S.E.M of three experiments. *PB0.05, **PB 0.001 respect to untreated cells.
[1] Steinberg D, Lewis A. Conner memorial lecture. Oxidative modification of LDL and atherogenesis. Circulation 1997;95:1062 – 71. [2] Esterbauer H, Ramos P. Chemistry and pathophysiology of oxidation of LDL. Rev Physiol Biochem Pharmacol 1995;127:31 – 64. [3] Hoppe G, Ravandi A, Herrera D, Kuksis A, Hoff HF. Oxidation products of cholesteryl linoleate are resistant to hydrolysis in macrophages, form complexes with proteins, and are present in human atherosclerotic lesions. J Lipid Res 1997;38:1347 –60. [4] Mu¨ ller K, Hardwick SJ, Marchant CE, et al. Cytotoxic and chemotactic potencies of several aldehydic components of oxidized low density lipoprotein for human monocyte-macrophages. FEBS Lett 1996;388:165 – 8. [5] Ruef J, Rao GN, Li F, Bode C, Patterson C, Bhatnagar A, Runge MS. Induction of rat aortic smooth muscle cell growth by lipid peroxidation product 4-hydroxy-2-nonenal. Circulation 1998;24:1071 – 8. [6] Keller JN, Mark RJ, Bruce AJ, et al. 4-HNE and aldehydic product of membrane lipid peroxidation, impairs glutamate transport and mitochondrial function in synaptosomes. Neuroscience 1997;80:685 – 96. [7] Steinbrecher UP, Lougheed M, Kwan WC, Dirks M. Recognition of oxidized low density lipoprotein by the scavenger receptor of macrophages results from derivatization of apolipoprotein by-products of fatty acid peroxidation. J Biol Chem 1989;264:15 216 – 5 223. [8] Esterbauer H. Cytotoxicity and genotoxicity of lipid-oxidation products. Am J Clin Nutr 1993;57:779S – 86S Supplementary. [9] Rajavashisth TB, Andalibi MC, Territo JA, Berliner M, Navad M, Fogelman AM, Lusis AJ. Induction of endothelial cell expression of granulocyte and macrophage colony-stimulating factors by modified low-density lipoproteins. Nature 1990;344:254 – 7. [10] Liu Y, Hulte´ n LM, Wiklund O. Macrophages isolated from human atherosclerotic plaques produce IL-8, and oxysterols may have a regulatory function for IL-8 production. Arterioscler Thromb Vasc Biol 1997;17:317 – 23. [11] Hamilton TA, Guoping MA, Chisolm GM. Oxidized low density lipoprotein suppresses the expression of Tumor Necrosis Factora mRNA in stimulated murine peritoneal macrophages. J Immunol 1990;144:2343 – 50. [12] Lipton BA, Parthasarathary S, Ord VA, Clinton SK, Libby P, Rosenfeld ME. Components of the protein fraction of oxidized low density lipoprotein stimulate interleukin-1 alpha production by rabbit arterial macrophage-derived foam cells. J Lipid Res 1995;36:2232 – 42. [13] Ohlsson BG, Englund MCO, Karlsson ALK, et al. Oxidized low density lipoprotein inhibits lipopolysaccharide-induced binding of nuclear factor-kB to DNA and the subsequent expression of Tumor Necrosis Factor-a and Interleukin-1b in macrophages. J Clin Invest 1996;98:78 – 89. [14] Hamilton TA, Major JA, Chilsom GM. The effects of oxidized low density lipoproteins on inducible mouse macrophage gene expression are gene and stimulus dependent. J Clin Invest 1995;95:2020 – 7.
J. Girona et al. / Atherosclerosis 158 (2001) 95–101 [15] Girona J, La Ville AE, Heras M, Olive´ S, Masana L. Oxidized lipoproteins including HDL and their lipid peroxidation products inhibit TNF-a secretion by THP-1 human macrophages. Free Rad Biol Med 1997;23:658 – 67. [16] Thomas CE, Jackson RL, Ohlweiler DF, Ku G. Multiple lipid oxidation products in low density lipoproteins induce interleukin-1 beta release from human blood mononuclear cells. J Lipid Res 1994;35:417 –27. [17] Auwerx J. The human leukemia cell line, THP-1: a multifaceted model for the study of monocyte-macrophage differentiation. Experientia 1991;47:22 –30. [18] Wang AM, Doyle MV, Mark DF. Quantitation of mRNA by the polymerase chain reaction. Proc Natl Acad Sci USA 1989;86:9717 – 21. [19] Parhami F, Fang ZT, Fogelman AM, Andalibi A, Territo MC, Berliner JA. Minimally modified low density lipoprotein-induced induced inflammatory responses in endothelial cells are mediated by cyclic adenosine monophosphate. J Clin Invest 1993;92:471 – 8. [20] Berliner JA, Navad M, Fogelman AM, et al. Atherosclerosis: basic mechanisms. Oxidation, inflammation, and genetics. Circulation 1995;91:2488 –96. [21] Liu-Wu Y, Hurt-Camejo E, Wiklund O. Lysophosphatidylcholine induces the production of IL-1b by human monocytes. Atherosclerosis 1998;137:351 – 7. [22] Parola M, Robino G, Marra F, et al. HNE interacts directly
.
[23]
[24]
[25]
[26]
[27]
[28] [29]
101
with JNK isoforms in human hepatic stellate cells. J Clin Invest 1998;102:1942 – 50. Ku G, Thomas CE, Akeson AL, Jackson RL. Induction of interleukin 1beta expression from human peripheral blood monocyte-derived macrophages by 9-hydroxyoctadecadienoic acid. J Biol Chem 1992;267:14 183 – 4 188. Fong LG, Fong TAT, Cooper AD. Inhibition of lipopolysaccharide-induced interleukin-1b mRNA expression in mouse macrophages by oxidized low density lipoprotein. J Lipid Res 1991;32:1899 – 910. Yla¨ -Herttuala S, Palinski W, Rosenfeld ME, et al. Evidence for the presence of oxidatively modified low density lipoprotein in atherosclerotic lesions of rabbit and man. J Clin Invest 1989;84:1086 – 95. Jovinge S, Ares MPS, Kallin B, Nilsson J. Human monocytes/ macrophages release TNF-a in response to ox-LDL. Arterioscler Thromb Vasc Biol 1996;16:1573 – 9. Page S, Fischer C, Baumgartner B, et al. 4-Hydroxynonenal prevents NF-kB activation and tumor necrosis factor expression by inhibiting IkB phosphorylation and subsequent proteolysis. J Biol Chem 1999;274:11 611 – 1 618. Jiang C, Ting AT, Seed B. PPAR-g agonists inhibit production of monocyte inflammatory cytokines. Nature 1998;391:82 –6. Nagy L, Tontonoz P, Alvarez J, Chen H, Evans R. Oxidized LDL regulates macrophages gene expression through ligand activation of PPAR-g. Cell 1998;93:229 – 40.
2,4-Decadienal downregulates TNF-a gene expression in THP-1 human macrophages Josefa Girona, Joan-Carles Vallve´, Josep Ribalta, Mercedes Heras, Sı´lvia Olive´, Lluı´s Masana * Unitat de Recerca de Lı´pids i Arteriosclerosi, Facultat de Medicina, Uni6ersitat Ro6ira i Virgili, C/Sant Llorenc¸ 21, 43201 Reus, Spain Received 6 July 2000; received in revised form 27 December 2000; accepted 3 January 2001
Abstract Oxidized lipoproteins inhibit TNF-a secretion by human THP-1 macrophages due, at least in part, to aldehydes derived from the oxidation of polyunsaturated fatty acids. This study extends these findings by investigating the effect of three aldehydes (2,4-decadienal (2,4-DDE), hexanal and 4-hydroxynonenal (4-HNE)) on TNF-a and IL-1b mRNA expression. The 2,4-DDE and 4-HNE showed considerable biological activity which induced cytotoxicity on THP-1 macrophages at concentration of 50 mM. Hexanal, on the other hand, had a lower cytotoxic capacity and concentration of 1000 mM was needed for the effect to be observed. Exposure of THP-1 macrophages to aldehydes for 24 h inhibited TNF-a mRNA expression but increased or did not affect IL-1b mRNA levels. The inhibitory action of 2,4-DDE was dose dependent and began at 5 mM (46%, P B 0.001). The effect of 4-HNE was less inhibitory than 4-DDE but only when cytotoxic concentrations were used (50 mM). Very high concentrations of hexanal (200 mM) were needed to inhibit TNF-a expression (23%, PB 0.001). This downregulation of TNF-a gene expression by 2,4-DDE was parallel to a lower protein production. These data indicate that low levels of 2,4-DDE may modulate inflammatory action by inhibiting TNF-a mRNA gene expression and that the biological activity of 2,4-DDE may be involved in the development of atherosclerosis. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Lipid peroxidation; Aldehydes; 2,4-Decadienal; THP-1 human macrophages; TNF-a; Gene expression; Atherosclerosis
1. Introduction There is increasing evidence to suggest that lipoprotein oxidation plays an important role in the pathogenesis of atherosclerosis [1]. Oxidation is one of the most likely ways of modifying low density lipoprotein (LDL) in vivo. However, oxidized LDL (ox-LDL) is a complex mixture of various components including lipid hydroperoxides, oxysterols and aldehydes [2]. Of these, some aldehydes diffuse from lipoproteins and induce biological effects by themselves. Core aldehydes are present in human atherosclerotic lesions [3]. Malondialdehyde (MDA), 4-HNE and hexanal are some of the aldehydes in ox-LDL and are chemotatic for human monocyte-macrophages [4]. 4-HNE induces vascular * Corresponding author. Tel.: + 34-977-759318; fax: +34-977759322. E-mail address: [email protected] (L. Masana).
smooth muscle cell growth by stimulating c-fos and c-jun expression [5] and can bind directly to membrane regulatory proteins [6]. The covalent binding of aldehydes with apolipoprotein B leads to the formation of immunogenic structures (4-HNE, MDA) that are recognized by the scavenger receptors of macrophages in ox-LDL [7]. This, in turn, leads to the formation of foam cells. Some aldehydes can also induce potent cytotoxic effects [8]. Oxidized lipoproteins can promote or suppress the expression of inflammatory cytokines in endothelial cells and mononuclear phagocytes [9–13] and it has been reported that suppression of TNF-a expression by ox-LDL is contained within the lipid fraction [14]. We have previously demonstrated that the fact that oxidized lipoproteins inhibit TNF-a secretion is partly due to aldehydes [15]. The effect of 2,4-DDE, which inhibits TNF-a and stimulates IL-1b secretion, is particularly strong. It also induces considerably more cytotoxicity than other aldehydes and hydroperoxides [16].
0021-9150/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 2 1 - 9 1 5 0 ( 0 1 ) 0 0 4 1 9 - 1
96
J. Girona et al. / Atherosclerosis 158 (2001) 95–101
Because of the role of these aldehydes might have in the pathogenesis of atherosclerosis, in this paper we discuss how 2,4-DDE and other aldehydes affect, on the mRNA level, the modulation of TNF-a and IL-1b in macrophages.
2. Materials and methods
2.1. Cell culture The human monocytic leukemia cell line THP-1 [17] was obtained from the American Type Culture Collection (ATCC c TIB 202) and maintained in RPMI1640 (Gibco-BRL) medium supplemented with heat-inactivated 10% FBS (Gibco-BRL, Myoclone Super Plus FBS, USA), 100 U/l penicillin and 100 mg/ml streptomycin. 2-mercaptoethanol was added to a final concentration of 0.5 mM and the cells were placed in a humidified incubator at 37°C and 5% CO2 until there were enough cells available for experiments. The cells were pelleted by centrifugation and plated at a final concentration of 1× 106 cells/ml in 12 multiwell dishes (Nalge Nunc Int., USA). Phorbol 12-myristate 13-acetate (PMA) at a final concentration of 50 ng/ml in dimethyl sulfoxide (DMSO) (final concentration 0.1%) was added to the medium to differentiate monocytes from macrophages [17]. During macrophage differentiation, TNF-a expression has a biphasic behaviour with a maximum at 2 h when cells begin to secrete protein (data not shown). After 72 h incubation with PMA, the cells were washed three times with PBS and fresh PMA free medium was added. Cells were used for the experiments after 2 days of incubation with complete medium and without PMA, in order to do the experiments in non-high activated cells. Basal levels of TNF-a in these cells were lower than after differentiation with PMA. Incubations were done in medium supplemented with heat-inactivated 10% FBS to avoid toxicity.
2.2. Cytotoxicity The amount of LDH released into the medium determined the cytotoxic effect and it was measured in a Cobas – Mira autoanalyzer (Roche, Switzerland) using an enzymatic method (Boehringer Mannheim, GmbH, Germany), optimized according to the recommendation of the Deutsche Gesellschaf for Klinische Chemie. This method is based on the direct, NADH2-coupled assay which uses pyruvate as the substrate. The absorbance was read at 340 nm. Results are expressed as LDH leakage (%), which represents the amount in the supernatant plus the cells as determined following cell lysis (1% triton). Cells were also visualized under phase-contrast microscopy so that morphological changes could be assessed.
2.3. Dose response experiments Macrophages were incubated with 2,4-DDE at concentrations of 0.05, 0.5, 1, 5, 10, 25 and 50 mM; hexanal at concentrations of 1, 25, 50, 100, 200, 500 and 1000 mM; and 4-HNE at concentrations of 0.5, 1, 5, 10, 20 and 50 mM for 24 h. A stock solution of each commercial pure aldehyde (Aldrich) was prepared. The final concentration of ethanol in the culture media was less than 0.5%. Cells incubated with vehicle alone are designated as untreated cells. After the incubations, the media were collected and centrifuged for 5 min at 900 rpm in order to discard non-adherent cells and cell debris. An aliquot of media was taken so that LDH could be measured immediately. Cells were used for total RNA extraction and IL-1b and TNF-a expression. For TNF-a protein studies macrophages were incubated with 2,4-DDE at concentrations of 0, 10, 20 and 50 mM for 24 h. Media were collected and stored at − 20°C and the TNF-a protein was determined within 1 month.
2.4. 2,4 DDE time response experiments Macrophages were incubated with 2,4-DDE (25 mM) and lipopolysaccharide (100 ng/ml) (E. coli LPS; serotype 026:B6, Sigma) for 0, 2, 6 and 24 h in triplicate. LPS was the positive control for cytokine expression. After the incubations, the media were discarded and the total RNA was extracted from the cells.
2.5. Analysis of TNF-h and IL-1i mRNA Total cellular RNA was isolated from the cells by the Ultraspec RNA isolation system (Biotecx Lab., Inc., USA). The oligonucleotide primers (Life Technologies, USA) for TNF-a were 5% CCT TGG TCT GGT AGG AGA CG 3% and 5% CAG AGG GAA GAG TTC CCC AG 3% and for IL-1b they were 5% TGG AGA ACA CCA CTT GTT GCT CCA 3% and 5% AAA CAG ATG AAG TGC TCC TTC CAG G 3%. These were the ones suggested by Wang et al. [18]. A semi-quantitative RT-PCR method was used to determine TNF-a and IL-1b mRNA using the SuperScript one-step RT-PCR system (Life Technologies, USA). Fifty nanogram of total RNA were used. cDNA synthesis and pre-denaturation were performed at 55°C for 30 min and then at 94°C for 2 min. For TNF-a, the PCR amplification consisted of first denaturing the DNA chains at 94°C for 30 s, then annealing them at 57°C for 30 s and finally extending them at 72°C for 30 s and 32 cycles. For IL-1b, the PCR amplification consisted of first denaturing the DNA chains at 94°C for 30 s, then annealing them at 55°C for 30 s and finally extending them at 72°C for 30 s and 36 cycles. There was a final
J. Girona et al. / Atherosclerosis 158 (2001) 95–101
extension at 72°C for 7 min. The b-actin gene was used as the loading control. All incubations were carried out in a Perkin-Elmer 2400 Thermal Cycler. The PCR products were separated on a 2% agarose gel. Ten microliter of each PCR mixture (25 ml) was used for electrophoresis which was run at 70 V for 45 min. The bands were visualized with ethidium bromide and the net intensity of each band was analyzed in the Kodak Digital Science 1D program. Results are expressed as the relative mRNA intensity and the mean cytokine/bactin net intensity ratio.
97
3.2. TNF-h and IL-1i mRNA expression studies Fig. 3 shows the effect of aldehydes on TNF-a and IL-1b mRNA levels. The dose-response experiments showed that either 2,4-DDE, 4-HNE or hexanal inhibited TNF-a mRNA expression after 24 h of incubation. In contrast, these aldehydes had no statistically significant effect on IL-1b mRNA levels at non-cytotoxic concentrations (Fig. 3). Among all the aldehydes tested, 2,4-DDE was the strongest inhibitor of TNF-a mRNA levels. Thus, addition of 2,4-DDE at concentration of 5
2.6. Determination of TNF-h and IL-1i protein secretion Cytokines were measured in the culture medium using commercial enzyme-linked immunosorbent assays (ELISA, Endogen, Inc., USA). The concentration of TNF-a was calculated as pg/ml using recombinant human TNF-a as standard at a concentration between 25.6 and 1000 pg/ml. The concentration of IL-1b was calculated as pg/ml using recombinant human IL-1b as standard at a concentration between 3.9 and 125 pg/ml. Blank values were obtained by analyzing a cell-free medium. Results are expressed as a percentage of those obtained with the blank.
2.7. Statistics The results are presented as means9 S.E.M. The statistical significance between two groups was determined by using the Student’s t-test. Various groups were compared by ANOVA and the statistical significance was calculated using the Bonferroni multiple comparison test. Statistical significance was set at a level of PB0.05. The Statistical Package for the Social Sciences (SPSS) software package version 8.0 was used for statistical analysis.
3. Results
3.1. Cytotoxicity e6aluation Fig. 1 shows the cytotoxic effects of 2,4-DDE, hexanal and 4-HNE on THP-1 macrophages. After 24 h of incubation with aldehydes, LDH release increased significantly with respect to the controls at a concentration of 50 mM for 2,4-DDE and 4-HNE and of 1000 mM for hexanal (P B 0.001), whereas lower concentrations of these aldehydes showed no cytotoxicity on macrophages. The cytotoxic effects were also confirmed at the same concentrations by the direct observation of morphological changes under phase-contrast microscopy (Fig. 2).
Fig. 1. Cytotoxicity of aldehydes. THP-1 macrophages were incubated with 0 – 50 mM of 2,4-DDE (A); 0 – 1000 mM of hexanal (B); 0 – 100 mM of 4-HNE (C), for 24 h. Results are expressed as LDH leakage (%) that represents the amount in the supernatant plus the cells as determined following cell lysis (1% triton). The data are shown as the mean9SEM of three experiments. *PB 0.001, compared to untreated cells.
98
J. Girona et al. / Atherosclerosis 158 (2001) 95–101
shows that non-cytotoxic concentrations did not modifiy IL-1b protein levels. We have also analysed by HPLC the content of 2,4-DDE in the medium after the incubation and we observed that 2,4-DDE remains in the medium when cells are incubated with 50 mM, suggesting that the effect observed was due to the direct action of free aldehyde (data not shown).
4. Discussion
Fig. 2. Phase-contrast microscopy showing the morphological changes of THP-1 macrophages after 24 h in absence (A) or presence (B) of 50 mM of 2,4-DDE, (X100).
mM significantly inhibited TNF-a mRNA levels by 46% (PB 0.001). This effect was greatest at 25 mM (88%, P B 0.001). 4-HNE had an inhibitory effect only when cytotoxic concentrations (50 mM) were used and the inhibition was lower than 2,4-DDE at the same concentration (32%, PB 0.001). Hexanal induced inhibition of TNF-a mRNA levels when very high concentrations were used in the experiments (]200 mM) and the percentage of inhibition was clearly lower (23%, PB 0.001) than that observed 2,4-DDE at 5 mM. Fig. 4 shows the results of the time-response experiments of 2,4-DDE. When compared to basal expression, 2,4-DDE at concentration of 25 mM induced a maximum inhibition effect on THP-1 macrophages at 2 h of incubation. A maximum inhibition was observed at 2 h (78%), with a recovery at 24 h (43%). This effect was completely different from that induced by LPS (100 ng/ml) used as a positive control in these experiments.
3.3. 2,4 -DDE suppresses the secretion of TNF-h Incubation of THP-1 macrophages with 2,4-DDE not only inhibited TNF-a mRNA levels but also reduced the secretion of the protein into the medium in a dose-dependent manner. This effect was observed at a concentration of 10 mM (33%) and reached statistical significance at 20 mM (68%, PB 0.05) (Fig. 5A). Fig. 5B
There is a great body of evidence to suggest that oxidized lipoproteins play an important role in the pathogenesis of atherosclerosis [1]. However the mechanisms involved in this process are not fully understood. Oxidized lipoproteins are taken up by macrophages that become foam cells. Minimally oxidized LDL also seems to participate in the endothelial activation and recruits inflammatory cells in the intima of the vessel wall [19]. Oxidized lipoproteins also participate in the initiation and propagation of the inflammatory process by modulating the expression of several genes involved in the arteriosclerotic process [20]. The oxidized lipoprotein is probably a mixture of particles with different degrees of oxidation. Furthermore, there are many different products derived from lipid peroxidation that have important biological actions as oxysterols derived from cholesterol oxidation, lysophosphatilcholine derived from phospholipid oxidation, hydroperoxides and aldehydes derived from fatty acid oxidation [21– 23]. These products can remain in the particle after the oxidative process but it is more likely that most of them will leave the lipoprotein and induce their biological effects away from the particle. It has also been demonstrated that the effect of oxidized lipoproteins on inflammation is variable and appears to depend, in part, on the extent of oxidation [16,24]. It is important to identify the molecules responsible for the biological effect of the oxidized lipoprotein. Previous reports have shown that extensively oxidized LDL and HDL can inhibit TNF-a secretion by macrophages [11,15]. This effect was mainly mediated by some aldehydes derived from the oxidation of polyunsaturated fatty acids, and 2,4-DDE in particular was seen to have a very powerful effect. The present study discusses whether 2,4-DDE acts at the genetic level and analyzes its effect on TNF-a and IL-1b expression in THP-1 human macrophages. At lower concentrations, the cytotoxic activity of 2,4-DDE was much stronger than hexanal and 4-HNE. At non-toxic concentrations, however, 2,4DDE clearly inhibited TNF-a mRNA expression, unlike the case with IL-1b. In the same system LPS increased the expression of the TNF-a gene. The inhibition by 2,4-DDE could be observed at concentrations of less than 1 mM although it reached statistical signifi-
J. Girona et al. / Atherosclerosis 158 (2001) 95–101
cance at 5 mM and its maximum effect was at 25 mM. Hexanal required cytotoxic concentrations that were 200 times higher than those for 2,4-DDE to induce the same level of inhibition. The 4-HNE also produced a small inhibitory effect at cytotoxic concentrations. The inhibition was maximum after 2 h of aldehyde incubation and after 24 h there was a partial recovery. Like the mRNA expression, the production of protein also decreased and at a concentration of 20 mM was finally reduced by 60%. Although there are no quantitative data on the concentration of aldehydes in the artery wall, their presence has been demonstrated by the presence of antibodies against these substances [25]. Our results strongly suggest that lipoprotein oxidation is something other than just lipoprotein modification. Many molecules derived from the lipid oxidation process are responsible for the effects independently of the lipoprotein modification. Exactly why in vitro oxidized lipoproteins inhibit TNF-a is not clear but probably depends on the degree of lipoprotein oxidation. It has
99
been observed that minimally oxidized LDL can increase the production of TNF-a [26], while extensively oxidized LDL suppresses it [11,15]. The mechanisms by which 2,4-DDE repress TNF-a gene expression are not known but it probably interacts with some transcription factors. A recently published report demonstrates that 4-HNE specifically inhibits the activation of NFkB [27]. It has also been shown that the interaction of oxysterols with NF-kB, and AP-1 inhibits the TNF-a gene [14]. An interesting hypothesis is that 2,4-DDE derived from fatty acid oxidation could interact with PPAR-g since PPAR-g agonists specifically inhibit TNF-a promoter-regulated gene expression [28]. It has also been demonstrated recently that products of PUFAs oxidation, 9-HODE and 13-HODE are endogenous activators and ligands of PPAR-g [29]. Our data show that the biological activity of 2,4-DDE may have some pathogenic implications in the development of atherosclerosis and therefore, it could be considered as a new therapeutical target.
Fig. 3. Dose-response of 2,4-DDE on the TNF-a and IL-1b gene expression by THP-1 macrophages. Cells were incubated with 0 – 50 mM of 2,4-DDE (A); 0 – 1000 mM of hexanal (B); and 0 –50 mM of 4-HNE (C), for 24 h. Total cellular RNA was isolated as described in the materials and methods section. Expression of TNF-a and IL-1b was evaluated by semiquantitative RT-PCR using the a-actin gene as control. Results are expressed as the percentage of the relative mRNA intensity with respect to untreated cells. Control values for 2,4-DDE, hexanal and 4-HNE were 1.239 0.08, 1.27 9 0.10 and 1.10 90.01 mRNA Intensity Ratio for TNF-a and 1.60 9 0.09, 1.47 9 0.01 and 1.33 90.05 mRNA Intensity Ratio for IL1-b, respectively. The data are shown as the mean 9 S.E.M of three experiments. *PB 0.05, **PB 0.001 compared to the untreated cells.
100
J. Girona et al. / Atherosclerosis 158 (2001) 95–101
Acknowledgements This work was supported by a grant from the Fondo de Investigacio´ n Sanitaria (FIS 98/0228).
References
Fig. 4. Time-course of 2,4-DDE and LPS on the TNF-a gene expression by THP-1 macrophages. Cells were incubated with 25 mM 2,4-DDE or 100 ng/ml LPS up to 24 h. Total cellular RNA was isolated as described in Section 2. Expression of TNF-a was evaluated by semiquantitative RT-PCR using the a-actin gene as control. Results are expressed as the percentage of the relative mRNA intensity with respect to the control for each time point. The data are shown as the mean 9S.E.M of two experiments.
Fig. 5. Effect of 2,4-DDE on cytokine secretion. Cells were incubated with 10, 20 and 50 mM of 2,4-DDE for 24 h. After incubation, media were collected and TNF-a (A) and IL-1b (B) was measured with an ELISA kit. Results are expressed as pg/ml. The data are shown as the mean 9 S.E.M of three experiments. *PB0.05, **PB 0.001 respect to untreated cells.
[1] Steinberg D, Lewis A. Conner memorial lecture. Oxidative modification of LDL and atherogenesis. Circulation 1997;95:1062 – 71. [2] Esterbauer H, Ramos P. Chemistry and pathophysiology of oxidation of LDL. Rev Physiol Biochem Pharmacol 1995;127:31 – 64. [3] Hoppe G, Ravandi A, Herrera D, Kuksis A, Hoff HF. Oxidation products of cholesteryl linoleate are resistant to hydrolysis in macrophages, form complexes with proteins, and are present in human atherosclerotic lesions. J Lipid Res 1997;38:1347 –60. [4] Mu¨ ller K, Hardwick SJ, Marchant CE, et al. Cytotoxic and chemotactic potencies of several aldehydic components of oxidized low density lipoprotein for human monocyte-macrophages. FEBS Lett 1996;388:165 – 8. [5] Ruef J, Rao GN, Li F, Bode C, Patterson C, Bhatnagar A, Runge MS. Induction of rat aortic smooth muscle cell growth by lipid peroxidation product 4-hydroxy-2-nonenal. Circulation 1998;24:1071 – 8. [6] Keller JN, Mark RJ, Bruce AJ, et al. 4-HNE and aldehydic product of membrane lipid peroxidation, impairs glutamate transport and mitochondrial function in synaptosomes. Neuroscience 1997;80:685 – 96. [7] Steinbrecher UP, Lougheed M, Kwan WC, Dirks M. Recognition of oxidized low density lipoprotein by the scavenger receptor of macrophages results from derivatization of apolipoprotein by-products of fatty acid peroxidation. J Biol Chem 1989;264:15 216 – 5 223. [8] Esterbauer H. Cytotoxicity and genotoxicity of lipid-oxidation products. Am J Clin Nutr 1993;57:779S – 86S Supplementary. [9] Rajavashisth TB, Andalibi MC, Territo JA, Berliner M, Navad M, Fogelman AM, Lusis AJ. Induction of endothelial cell expression of granulocyte and macrophage colony-stimulating factors by modified low-density lipoproteins. Nature 1990;344:254 – 7. [10] Liu Y, Hulte´ n LM, Wiklund O. Macrophages isolated from human atherosclerotic plaques produce IL-8, and oxysterols may have a regulatory function for IL-8 production. Arterioscler Thromb Vasc Biol 1997;17:317 – 23. [11] Hamilton TA, Guoping MA, Chisolm GM. Oxidized low density lipoprotein suppresses the expression of Tumor Necrosis Factora mRNA in stimulated murine peritoneal macrophages. J Immunol 1990;144:2343 – 50. [12] Lipton BA, Parthasarathary S, Ord VA, Clinton SK, Libby P, Rosenfeld ME. Components of the protein fraction of oxidized low density lipoprotein stimulate interleukin-1 alpha production by rabbit arterial macrophage-derived foam cells. J Lipid Res 1995;36:2232 – 42. [13] Ohlsson BG, Englund MCO, Karlsson ALK, et al. Oxidized low density lipoprotein inhibits lipopolysaccharide-induced binding of nuclear factor-kB to DNA and the subsequent expression of Tumor Necrosis Factor-a and Interleukin-1b in macrophages. J Clin Invest 1996;98:78 – 89. [14] Hamilton TA, Major JA, Chilsom GM. The effects of oxidized low density lipoproteins on inducible mouse macrophage gene expression are gene and stimulus dependent. J Clin Invest 1995;95:2020 – 7.
J. Girona et al. / Atherosclerosis 158 (2001) 95–101 [15] Girona J, La Ville AE, Heras M, Olive´ S, Masana L. Oxidized lipoproteins including HDL and their lipid peroxidation products inhibit TNF-a secretion by THP-1 human macrophages. Free Rad Biol Med 1997;23:658 – 67. [16] Thomas CE, Jackson RL, Ohlweiler DF, Ku G. Multiple lipid oxidation products in low density lipoproteins induce interleukin-1 beta release from human blood mononuclear cells. J Lipid Res 1994;35:417 –27. [17] Auwerx J. The human leukemia cell line, THP-1: a multifaceted model for the study of monocyte-macrophage differentiation. Experientia 1991;47:22 –30. [18] Wang AM, Doyle MV, Mark DF. Quantitation of mRNA by the polymerase chain reaction. Proc Natl Acad Sci USA 1989;86:9717 – 21. [19] Parhami F, Fang ZT, Fogelman AM, Andalibi A, Territo MC, Berliner JA. Minimally modified low density lipoprotein-induced induced inflammatory responses in endothelial cells are mediated by cyclic adenosine monophosphate. J Clin Invest 1993;92:471 – 8. [20] Berliner JA, Navad M, Fogelman AM, et al. Atherosclerosis: basic mechanisms. Oxidation, inflammation, and genetics. Circulation 1995;91:2488 –96. [21] Liu-Wu Y, Hurt-Camejo E, Wiklund O. Lysophosphatidylcholine induces the production of IL-1b by human monocytes. Atherosclerosis 1998;137:351 – 7. [22] Parola M, Robino G, Marra F, et al. HNE interacts directly
.
[23]
[24]
[25]
[26]
[27]
[28] [29]
101
with JNK isoforms in human hepatic stellate cells. J Clin Invest 1998;102:1942 – 50. Ku G, Thomas CE, Akeson AL, Jackson RL. Induction of interleukin 1beta expression from human peripheral blood monocyte-derived macrophages by 9-hydroxyoctadecadienoic acid. J Biol Chem 1992;267:14 183 – 4 188. Fong LG, Fong TAT, Cooper AD. Inhibition of lipopolysaccharide-induced interleukin-1b mRNA expression in mouse macrophages by oxidized low density lipoprotein. J Lipid Res 1991;32:1899 – 910. Yla¨ -Herttuala S, Palinski W, Rosenfeld ME, et al. Evidence for the presence of oxidatively modified low density lipoprotein in atherosclerotic lesions of rabbit and man. J Clin Invest 1989;84:1086 – 95. Jovinge S, Ares MPS, Kallin B, Nilsson J. Human monocytes/ macrophages release TNF-a in response to ox-LDL. Arterioscler Thromb Vasc Biol 1996;16:1573 – 9. Page S, Fischer C, Baumgartner B, et al. 4-Hydroxynonenal prevents NF-kB activation and tumor necrosis factor expression by inhibiting IkB phosphorylation and subsequent proteolysis. J Biol Chem 1999;274:11 611 – 1 618. Jiang C, Ting AT, Seed B. PPAR-g agonists inhibit production of monocyte inflammatory cytokines. Nature 1998;391:82 –6. Nagy L, Tontonoz P, Alvarez J, Chen H, Evans R. Oxidized LDL regulates macrophages gene expression through ligand activation of PPAR-g. Cell 1998;93:229 – 40.