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Cell Biology International (2007) 31, 14561461 (Printed in Great Britain)
Interleukin-10 inhibits the down-regulation of ATP binding cassette transporter A1 by tumour necrosis factor-alpha in THP-1 macrophage-derived foam cells
Chun‑li Mei, Zhi‑jian Chen*, Yu‑hua Liao, Yan‑fu Wang, Hong‑yu Peng and Yu Chen
Department of Cardiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1277 Jie-Fang Avenue, Wuhan, Hubei Province 430022, China
It is suggested that cholesterol efflux mediated by ATP binding cassette transporter A1 (ABCA1) plays an important role in anti-atherogenesis. However, the effects of inflammatory cytokines on ABCA1 expression and cholesterol accumulation in foam cells are little known. This study investigates the effects of tumour necrosis factor-alpha (TNF-α) and interleukin-10 (IL-10) on ABCA1 expression and cholesterol content in THP-1 macrophage-derived foam cells. ABCA1mRNA and protein levels were determined by RT-PCR and Western blot, respectively. The total cholesterol content in THP-1 macrophage-derived foam cells was detected by the zymochemistry method. Results revealed that TNF-α could increase cholesterol content by down-regulating ABCA1 expression in a time-dependent manner in THP-1 macrophage-derived foam cells, which may contribute to its pro-atherosclerotic effect. In addition IL-10 time-dependently decreased cholesterol accumulation by up-regulating ABCA1 expression and inhibited the down-regulation of ABCA1 by TNF-α in THP-1 macrophage-derived foam cells, which may be one of the mechanisms of IL-10 contributing to its anti-atherosclerotic action.
Keywords: ATP binding cassette transporter A1, Cholesterol content, Tumour necrosis factor-alpha, Interleukin-10, THP-1 macrophage-derived foam cells.
*Corresponding author. Tel./fax: +86 27 8572 7340.
In past decades, high density lipoprotein (HDL) has been proposed to decrease atherosclerosis mainly by reverse cholesterol transport (RCT), a process by which HDL carries excess cholesterol from peripheral tissues and cells, including foam cells, back to the liver for removal from the body (Von Eckardstein et al., 2001). A major breakthrough in the understanding of the mechanism of RCT came with the discovery of the ATP binding cassette transporter A1 (ABCA1) as the molecular defect in Tangier disease (TD), a rare genetic disease characterised by the accumulation of macrophage foam cells in various tissues, severe HDL deficiency, and prevalent atherosclerosis (Oram, 2000). ABCA1 belongs to the ATP binding cassette (ABC) transporter superfamily. It promotes efflux of free cholesterol and phospholipids from cellular membranes to apolipoprotein A-I (apoA-I) and initiates the formation of nascent HDL particles (Wang, 2003; Brewer et al., 2004). ABCA1 is widely expressed in the liver, kidney, adrenal gland, intestine and central nervous system (CNS) (Lawn et al., 2001). It is also prominent in the macrophage foam cells of atherosclerotic lesions. It has been suggested that cholesterol efflux mediated by ABCA1 plays an important role in anti-atherogenesis. Bone marrow transplantation studies have shown that the over-expression of ABCA1 in macrophage foam cells reduces diet-induced atherosclerosis in different mouse models (Aiello et al., 2002; Singaraja et al., 2002).
Atherosclerosis represents an inflammatory reaction in the arterial wall, initiated by the retention of lipoprotein lipids (Libby, 2002). A range of inflammatory cytokines have been demonstrated to be involved in atherogenesis, some with pro-atherogenic properties while others have anti-atherogenic properties. One of the most noticeable pro-inflammatory cytokines is tumour necrosis factor-alpha (TNF-α), which is active inatherosclerotic plaques (Rus et al., 1991). In vitro and in vivo studies have shown that TNF-α signaling is pro-atherogenic (Rus et al., 1991; Young et al., 2002). In contrast to TNF-α, interleukin-10 (IL-10) has pronounced immunosuppressive and anti-inflammatory effects. IL-10 deficiency aggravated and IL-10 over-expression attenuated experimental atherosclerosis (Potteaux et al., 2004; Namiki et al., 2004; Caligiuri et al., 2003). Rubic and Lorenz (2006) reported that IL-10 attenuated cholesterol accumulation by down-regulating CD36 and up-regulating ABCA1 expression in macrophages, indicating that inflammatory cytokines could regulate macrophage cholesterol homeostasis. However, these observations have provided little insight into understanding the relation between inflammatory cytokines and cholesterol accumulation in foam cells. We investigated the effects of TNF-α and IL-10 on ABCA1 expression and cholesterol content in THP-1 macrophage-derived foam cells.
2 Materials and methods
The human monocytes line THP-1 was obtained from Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. Trizol reagent and RPMI1640 powder were purchased from Gibco (Grand Island, NY). The RT-PCR kit was purchased from TaKaKa (Beijing, China). RNA extraction materials were from Qiagen Ltd (West Sussex, UK). Western blot consumables were purchased from Invitrogen (Carlsbad, CA, USA). Fetal bovine serum (FBS), bovine serum albumin (BSA), TNF-α, IL-10, apolipoprotein A-I (apoA-I) and all other chemicals were purchased from Sigma (St. Louis, MO, USA). Goat polyclonal anti-ABCA1, and horseradish peroxidase (HRP)-conjugated second antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
2.2 Low density lipoprotein (LDL) isolation and oxidization
Large amounts of plasma LDL were isolated by a two-step ultracentrifugation as described (Wang et al., 1995). LDL (d
The isolated LDL was oxidized with 0.01
2.3 Cell culture and foam cell model
THP-1 cells were seeded into six-well plates at 1
2.4 Electron microscopy
The cells were harvested and then fixed by 3% glutaral for 3
2.5 Pre-incubation phase
THP-1 foam cells were washed with PBS, and incubated in serum-free RPMI1640 medium containing 0.3% BSA and in the presence or absence of TNF-α (10
2.6 RT-PCR for ABCA1mRNA expression
Total RNA was isolated and purified by Trizol reagent kit, according to the manufacturer's protocol. RT-PCRs were carried out in 20
Primers used in the RT-PCR analysis
2.7 Western blot for ABCA1 protein level
Cells were washed with pre-cooling PBS, centrifuged at 4
2.8 Measurement of cellular total cholesterol
The cellular total cholesterol content was measured by zymochemistry method as described (Gamble et al., 1978). After pre-incubation phase at various times from 0 to 48
2.9 Statistical analysis
The experimental data were expressed as means
3.1 Formation of THP-1 macrophage-derived foam cells
As shown in Fig. 1, compared with macrophages (Fig. 1A), many lipid-filled vacuoles appeared in the cytoplasm of ox-LDL-treated THP-1 macrophages (Fig. 1B). The results of electron microscopy indicate the formation of THP-1 macrophage-derived foam cells in morphology.
Formation of THP-1 macrophage-derived foam cells. (A) ox-LDL-untreated THP-1 macrophages were observed by electron microscopy. (B) ox-LDL-treated THP-1 macrophages were observed by electron microscopy. Black arrow indicates lipid-filled vacuoles in cytoplasm. Magnification ×80000.
3.2 Effects of TNF-α and IL-10 on ABCA1 expression in THP-1 macrophage-derived foam cells
RT-PCR revealed that TNF-α time-dependently down-regulated ABCA1mRNA expression in THP-1 macrophage-derived foam cells compared with control group (P
Effects of TNF-α and IL-10 on ABCA1 expression in THP-1 macrophage-derived foam cells. THP-1 macrophage-derived foam cells were incubated with TNF-α (10
In similar experiments, we investigated the levels of ABCA1 protein, as shown by Western blot as a band of ≈220
3.3 Effects of TNF-α and IL-10 on the total cholesterol content in THP-1 macrophage-derived foam cells
To study the effects of TNF-α and IL-10 on ABCA1-mediated cholesterol efflux, we investigated the total cholesterol content in THP-1 macrophage-derived foam cells (Fig. 3). TNF-α increased the total cholesterol content at 12
Effects of TNF-α and IL-10 on the total cholesterol content in THP-1 macrophage-derived foam cells. THP-1 macrophage-derived foam cells were incubated with TNF-α (10
Both clinical and biochemical evidence strongly suggest that the development of atherosclerotic lesions can be accelerated by local actions of inflammatory cytokines on endothelial cells (Young et al., 2002; Hansson, 2005). TNF-α is a key pro-inflammatory cytokine in the inflammatory process of atherosclerosis. It has been localised in atheromatous plaques, which it is thought to contribute to the development and progression of atherosclerosis by augmenting the local inflammatory response and altering lipid homeostasis (Rus et al., 1991; Napolitano and Bravo, 2005; Vogel et al., 2004). Previous studies have shown that pro-inflammatory cytokines promote lipid accumulation in human mesangial cell by inducing type A scavenger receptor (Francone et al., 2005).
This study has demonstrated that TNF-α inhibits ABCA1mRNA and protein levels in a time-dependent manner in THP-1 macrophage-derived foam cells. On the functional level, the down-regulation of ABCA1 by TNF-α was shown to increase cellular cholesterol accumulation. It provides a plausible mechanism by which TNF-α might impair cellular cholesterol efflux to lipid-free apoA-I by inhibiting ABCA1 expression. Inhibition of the ABCA1 pathway by TNF-α might in part explain the clinical observation that patients with an activated inflammatory response have low circulating levels of HDL in plasma. In fact, the levels of TNF-α are independent predicators of atherosclerotic cardiovascular events in older persons (Skoog et al., 2002). According to the recent data (Panousis and Zuckeman, 2000), the pro-inflammatory lymphokine gamma interferon is capable to down-regulate ABCA1 expression in macrophage-derived foam cells. Thus, our results are similar to previous studies, indicating pro-inflammatory cytokines have new pro-atherosclerotic effect by inhibiting ABCA1 expression and increasing cholesterol accumulation.
Importantly, we demonstrated that anti-inflammatory cytokine IL-10 induced ABCA1 expression and increased cholesterol efflux in a time-dependent manner in THP-1 macrophage-derived foam cells leading to the decrease of cholesterol content of foam cells. Moreover, we observed that the down-regulation of ABCA1 expression by TNF-α was time-dependently inhibited by pre-incubation with IL-10. Meantime, IL-10 also time-dependently attenuated the increment of cellular cholesterol content by TNF-α. These results suggest that IL-10 signaling may have a protective role in preventing cholesterol accumulation and atherogenesis by inducing ABCA1 expression and cholesterol efflux. The negative interaction of the two inflammatory cytokines on ABCA1 and cellular cholesterol content may be in part explained by the inhibition of activation of TNF-α by IL-10. IL-10 is a pleiotropic anti-inflammatory cytokine produced by many cell types including lymphocytes, mass cells and macrophages (Potteaux et al., 2004; Tedgui and Mallat, 2001; Mallat et al., 1999). Clinical trials as well as experimental studies have shown that IL-10 has anti-atherogenic effect (Potteaux et al., 2004; Namiki et al., 2004). Even in animal models such as apolipoprotein E (apoE) or LDL-receptor knockout mice, IL-10 profoundly reduced the lipid accumulation in the vessel wall (Potteaux et al., 2004; Pinderski et al., 2002). However, IL-10 cannot reverse the down-regulation of ABCA1 expression by TNF-α, indicating that inflammatory response cannot be a sole factor in regulating ABCA1 expression. Thus, unknown intermediated factors involved in regulating ABCA1 expression need to be identified further.
Gerbod-Giannone et al. (2006) reported that TNF-α up-regulated ABCA1 expression in macrophage and in phagocytes ingesting apoptotic cells. It is not clear whether the discrepancy is due to different cell types, species specificity or other unknown factors. Although Rubic and Lorenz (2006) reported that IL-10 up-regulated ABCA1 expression by liver X receptor (LXR) pathways in macrophages, the question remains as to the mechanism by which TNF-α and IL-10 regulate ABCA1 expression in foam cells. Nuclear factor-kappa B (NF-κB) is a key nuclear transcription factor, which plays an important role in regulation of a variety of genes involved in the inflammatory response, whose activation has been linked to the onset of atherosclerosis (Brand et al., 1996). TNF-α promoted the activation of NF-κB, while IL-10 inhibited the activation of NF-κB (Van Oostrom et al., 2004; Schottelius et al., 1999). We, therefore, also studied the effects of the activation of NF-κB on ABCA1 expression in THP-1 macrophage-derived foam cells. Our results indicated that the effects of TNF-α and IL-10 on ABCA1 expression and cholesterol accumulation in foam cells could be involved in the activation of NF-κB (data not shown).
In summary, this study provides new insights into the possible role of inflammatory cytokines in atherosclerosis. TNF-α could increase cholesterol accumulation by down-regulating ABCA1 expression in THP-1 macrophage-derived foam cells, which may contribute to its pro-atherosclerotic effect. IL-10 decreased cholesterol overloading by up-regulating ABCA1 expression and inhibited the effects of TNF-α in THP-1 macrophage-derived foam cells, which may be one of the mechanisms of IL-10 contributing to its anti-atherosclerotic action. Considering the critical role of the ABCA1 transporter in the regulation of cholesterol efflux and HDL metabolism, further investigation of the mechanisms underlying the effects of inflammatory cytokines on ABCA1 expression is important for a better understanding of inflammation and atherogenesis. Alternatively, the potential benefits from the anti-inflammatory process in atherosclerosis also deserve further evaluation.
The study was supported by Institute of Cardiovascular, Union Hospital, Wuhan, China. We acknowledge the assistance and excellent technical advice provided by Dr. Heping Guo (Institute of Cardiology, Union Hospital, Wuhan, China).
Aiello RJ, Brees, D, Bourassa, PA, Royer, L, Lindesy, S, Coskran, T. Increased atherosclerosis in hyperlipidemic mice with inactivation of ABCA1 in macrophages. Arterioscler Thromb Vasc Biol 2002:22:630-7
Brewer HB, Remaley, AT, Neufeld, EB, Basso, F, Joyce, C. Regulation of plasma high-density lipoprotein levels by the ABCA1 transporter and the emerging role of high-density lipoprotein in the treatment of cardiovascular disease. Arterioscler Thromb Vasc Biol 2004:24:1755-60
Brand K, Page, S, Rogler, G, Bartsch, A, Brandl, R, Knuechel, R. Activated transcription factor nuclear factor-kappa B is present in the atherosclerotic lesions. J Clin Invest 1996:97:1715-22
Caligiuri G, Rudling, M, Ollivier, V, Jacob, MP, Michel, JB, Hansson, GK. Interleukin-10 deficiency increases atherosclerosis, thrombosis, and low-density lipoprotein in apolipoprotein E knockout mice. Mol Med 2003:9:10-7
Francone OL, Royer, L, Boucher, G, Haghpassand, M, Freeman, A, Brees, D. Increased cholesterol deposition, expression of scavenger receptors, and response to chemotactic factors in Abca1-deficient macrophages. Arterioscler Thromb Vasc Biol 2005:25:1198-205
Gamble W, Vaughan, M, Kruth, HS, Avigan, J. Procedure for determination of free and total cholesterol in micro- or nanogram amounts suitable for studies with cultured cells. J Lipid Res 1978:19:1068-70
Gerbod-Giannone MC, Li, Y, Holleboom, A, Han, S, Hsu, LC, Tabas, I. TNF alpha induces ABCA1 through NF-kappa B in macrophages and in phagocytes ingesting apoptotic cells. Proc Natl Acad Sci U S A 2006:103:3112-7
Lawn RM, Wade, DP, Couse, TL, Wilcox, JN. Localization of human ATP-binding cassette transporter1 (ABCA1) in normal and atherosclerosis tissue. Aterioscler Thromb Vasc Biol 2001:21:378-85
Namiki M, Kawashima, S, Yamashita, T, Ozaki, M, Sakoda, T, Inoue, N. Intramuscular gene transfer of interleukin-10 cDNA reduces atherosclerosis in apolipoprotein E-knockout mice. Atherosclerosis 2004:172:21-9
Potteaux S, Esposito, B, van Oostrom, O, Brun, V, Ardouin, P, Groux, H. Leukocyte-derived interleukin 10 is required for protection against atherosclerosis in low-density lipoprotein receptor knockout mice. Arterioscler Thromb Vasc Biol 2004:24:1474-8
Panousis CG, Zuckeman, SH. Interferon gamma induces down regulation of Tangier disease gene (ATP binding cassette transporter1) in macrophage derived foam cells. Arteroscler Thromb Vasc Biol 2000:20:1565-72
Pinderski LJ, Fischbein, MP, Subbanagounder, G, Fishbein, MC, Kubo, N, Cheroutre, H. Overexpression of interleukin-10 by activated T lymphocytes inhibits atherosclerosis in LDL receptor-deficient mice by altering lymphocyte and macrophage phenotypes. Circ Res 2002:90:1064-71
Rubic T, Lorenz, RL. Downregulated CD36 and oxLDL uptake and stimulated ABCA1/G1 and cholesterol efflux as anti-atherosclerotic mechanisms of interleukin-10. Cardiovasc Res 2006:69:527-35
Scaccini C, Chiesa, G, Jialal, I. A critical assessment of the effects of aminoguanidine and ascorbate on the oxidative modification of LDL: evidence of interference with some assays of lipoprotein oxidation by aminoguanidine. J Lipid Res 1994:35:1085-92
Schottelius AJG, Mayo, MW, Sartor, RB, Baldwin, AS. Interleukin-10 signaling blocks inhibitor of κB kinase activity and nuclear factor κB DNA binding. J Biol Chem 1999:274:31868-74
Van Oostrom AJ, van Wijk, J, Cabezas, MC. Lipaemia. Inflammation and atherosclerosis: novel opportunities in the understanding and treatment of atherosclerosis. Drugs 2004:64:19-41
Von Eckardstein A, Nifer, JR, Assmann, G. High density lipoprotein and arteriosclerosis role of cholesterol efflux and reverse cholesterol transport. Arterioscler Thromb Vasc Biol 2001:21:13-27
Vogel CF, Sciullo, E, Matsumura, F. Activation of inflammatory mediators and potential role of ah-receptor ligands in foam cell formation. Cardiovasc Toxicol 2004:4:363-73
Wang C, Zong, Y, Wu, W. Rapid isolation of large amount of plasma VLDL and LDL by a two step ultracentrifugation. Acta Univ Med Tongji 1995:24:169-71
Received 12 April 2007/10 May 2007; accepted 12 June 2007doi:10.1016/j.cellbi.2007.06.009