Human Monocytes/Macrophages Release TNF-α in Response to Ox-LDL
The uptake of oxidatively modified low density lipoprotein (Ox-LDL) by intimal macrophages is believed to play a key role in the development of atherosclerosis. The present study demonstrates that Ox-LDL in low concentrations activates monocyte/macrophage release of factors that stimulate smooth muscle cell growth, whereas higher concentrations are inhibitory. Exposure of monocytes/macrophages to 8 μg/mL Ox-LDL increased expression of tumor necrosis factor-α (TNF-α) mRNA but had no effect on interleukin-1β, platelet-derived growth factor B and heparin-binding epidermal growth factor–like mitogen mRNA levels. Ox-LDL also stimulated monocyte/macrophage release of TNF-α in a dose-dependent manner, with maximal effect at an LDL concentration of 8 μg/mL. Addition of TNF-α–blocking antibodies to conditioned medium from monocytes/macrophages already exposed to Ox-LDL reduced mitogenic activity by 44.7±8.4% (P<.005). Stimulation of TNF-α release by Ox-LDL was associated with activation of transcription factor AP-1, whereas the activity of transcription factor nuclear factor-κB remained unchanged. These findings suggest that enhanced secretion of TNF-α by macrophages exposed to Ox-LDL may be involved in the formation of atherosclerotic lesions.
- Received September 1, 1995.
- Revision received March 22, 1996.
Hypercholesterolemia is one of the most prominent risk factors for atherosclerosis.1 2 3 The biological mechanisms whereby lipids induce the vascular inflammation and fibrosis that characterize atherosclerosis remain largely unknown, but recent findings have indicated that oxidative modification of LDL may play a key role in this process.4 LDL oxidation is believed to occur as LDL particles penetrate the arterial intima, become trapped by intimal glycosaminoglycans, and are exposed to free radicals released by surrounding cells.5 6 7 Oxidative modification of LDL results in its uptake by macrophage scavenger receptors and the formation of cholesterol-loaded macrophage foam cells,8 which constitute a major part of both early and more advanced lesions.8 9 10 11 LDL oxidation is associated with the formation of a number of highly reactive molecules, such as lipid peroxides, lysoPC, oxysterols, and aldehydes,12 which by interacting with surrounding cells may cause nonspecific activation at lower concentrations or cell death at higher concentrations.13 Several in vitro studies have suggested that low concentrations of fully Ox-LDL and/or higher concentrations of minimally modified LDL have proinflammatory effects, including stimulation of leukocyte binding to endothelial cells,14 induction of T-lymphocyte proliferation,15 and release of leukocyte chemoattractants.16
In hypercholesterolemic animal models of atherosclerosis, intimal accumulation of cholesterol-loaded macrophages is followed by the development of raised fibromuscular plaques.17 Accordingly, it has been proposed that macrophages play a role in the activation of intimal SMC growth, which is responsible for this transition.11 Indeed, monocytes/macrophages secrete several factors that activate SMC growth, such as PDGF-BB,18 19 IL-1β,20 TNF-α,21 and HB-EGF.22 Previous studies have shown that exposure of cultured macrophages to Ox-LDL at concentrations ≥15 μg/mL inhibits expression of these growth factors.23 24 The present study confirms these findings but also demonstrates that Ox-LDL at a lower concentration interval activates monocytes/macrophages to release factors that stimulate SMC growth and that most of this activity is due to increased release of TNF-α.
Venous blood was drawn from healthy, fasting donors into evacuated tubes (Vacutainer, Becton Dickinson) containing 1 mg/mL EDTA. LDL was prepared essentially as described by Redgrave and Carlsson,25 with an EDTA concentration of 10 μmol/L. The total protein content was determined according to the method of Lowry et al.26 Oxidation was performed by incubating 200 μg/mL LDL in 5 μmol/L CuSO4 in PBS at 37°C for 18 hours and confirmed by agarose gel electrophoresis. EDTA was removed from LDL by filtration on Econo-Pac 10DG columns (Bio-Rad) before oxidation or immediately before addition to the cell culture medium for control LDL. Fresh preparations of LDL were used for each experiment. Acetylation of LDL was performed as described by Basu et al.27
SMCs were isolated from the media of saphenous vein grafts. The specimens were carefully dissected, cut into small pieces, and allowed to become attached to six-well multiplates during a 15-minute drying period. The explants were then cultivated in F-12/DMEM (GIBCO BRL) containing 10% newborn calf serum (GIBCO BRL), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C in an atmosphere of 5% CO2 in air. Cells began migrating from the explants within 1 to 2 weeks and reached confluence within another 2 weeks. Secondary cultures were established by trypsinization and seeding of the cells in 75-cm2 culture flasks. The purity of SMC populations was determined by the presence of smooth muscle–specific α-actin immunoreactivity with the HHF-35 antibody.28 SMCs used for analysis of DNA synthesis were seeded in 96-well plates at 10 000 cells per well. Subconfluent cultures were growth arrested by incubation in F-12 medium supplemented with antibiotics and 1% plasma-derived serum for 48 hours. Experiments were performed on cells cultured for up to 10 passages.
Mononuclear leukocytes were obtained from buffy coats as described.29 Monocytes were isolated by adherence to plastic dishes in RPMI-1640 with 24 mmol/L NaHCO3, 25 mmol/L HEPES, 100 U/mL penicillin, 100 μg/mL streptomycin, 1 mmol/L sodium pyruvate, 4 mmol/L l-glutamine, heat-inactivated 10% fetal calf serum (GIBCO BRL), and 10% pooled human serum (GIBCO BRL). After 2 hours the cells were washed four times in Dulbecco's PBS. More than 90% of the adherent cell population was CD14-positive according to FACS analysis (Becton Dickinson). These cells represent monocytes in an early stage of macrophage differentiation and are thus referred to as monocytes/macrophages in the text.
Analysis of Macrophage-CM for Growth Factor Activity for SMC, IL-1β, and TNF-α Levels
Monocytes/macrophages were exposed to native or Ox-LDL in serum-free medium with 0.1% low-endotoxin BSA for 12 hours. Endotoxin levels in LDL preparations and CM as determined by the Limulus assay (Sigma) were <0.5 ng/mL and did not differ between native and Ox-LDL media. The CM was diluted 1:1 (vol/vol) with fresh F-12 medium supplemented with antibiotics and 2% plasma-derived serum. [3H]thymidine (Amersham) was added to each sample to yield a final concentration of 74 kBq/mL. Serum-starved SMCs were then incubated with conditioned or control medium for 48 hours, and [3H]thymidine uptake into the macromolecular material was determined by liquid scintillation counting as described previously.30
For ELISA analysis of IL-1β and TNF-α levels, 1.5 mL of CM was centrifuged for 10 minutes at 400g. Aliquots (200 μL) from three independent samples of supernatant were then added to high-sensitivity human TNF-α (R&D Systems) or IL-1β (Amersham) immunoassay plates. The plates were processed according to the manufacturers' protocols and read at 450 nm.
Blocking of TNF-α in CM
To block TNF-α activity in monocyte/macrophage–CM, a blocking IgG antibody (R&D Systems) against TNF-α was used at a concentration of 0.05 μg/mL. The medium was preincubated with the antibody for 1 hour at 37°C before the medium was added to the SMCs. Specificity of the blocking reaction was tested with an irrelevant mouse myeloma IgG (clone No. MOPC-195, Immunotech SA) that was applied in the same manner as the TNF-α antibody.
For analysis of mRNA levels, freshly isolated monocytes/macrophages were grown overnight in serum-containing medium as described above. The cells were then washed in Dulbecco's PBS and exposed to native or Ox-LDL in serum-free medium supplemented with 0.1% BSA. RNA was isolated as described by Chirgwin et al.31 Northern blotting and hybridization on DuPont-NEN Sorb Plus nylon membranes were performed according to the manufacturer's protocol (NEN Research Products). Blots were hybridized with 106 disintegrations per minute (dpm)/mL [α-32P]dCTP (Amersham) –labeled cDNA probes for TNF-α (Chiron Corp), PDGF-B (generously provided by Dr Christer Betsholtz, Gothenburg, Sweden), and IL-1β (generously provided by Dr Peter Libby, Boston, Mass).32 33 A 1-kb probe corresponding to bases 1049 to 2048 of the HB-EGF cDNA22 was prepared by PCR amplification, and γ-32P–labeled oligonucleotide for γ-actin (5′-ATG CCG GAG CCA TTG TCA ATG ACC AGC GCG GCG-3′; SGS AB) was used as the loading control.
Monocytes/macrophages were grown overnight in serum-containing medium and washed with Dulbecco's PBS as described for Northern blotting. Experiments were performed in serum-free medium with 0.1% BSA as described above. Nuclear extracts were prepared as described by Alksnis et al.34 EMSA was performed as described by Schütze et al.35 The probes were 32P-labeled double-stranded synthetic oligonucleotides (Santa Cruz Biotechnology) containing consensus binding sequences for transcription factors AP-1 (5′-CGC TTG ATG ACT CAG CCG GAA-3′) or NF-κB (5′-AGT TGA GGG GAC TTT CCC AGG C-3′). Specificity of the protein-DNA complexes was verified by competition assays with excess unlabeled oligonucleotides and oligonucleotides containing a mutated binding site. The mutant oligonucleotides used were 5′-CGC TTG ATG ACT TGG CCG GAA-3′ for AP-1 and 5′-AGT TGA GGC GAC TTT CCC AGG C-3′ for NF-κB (mutation sites underlined). Immunoreactivity of specific complexes was tested with rabbit polyclonal antibodies (Santa Cruz Biotechnology) against c-Jun, NF-κB1 (p50), and RelA (p65).
Values are given as mean±SEM. Between-group analyses were performed with an unpaired t test. A value of P<.05 was considered significant.
Release of SMC Mitogenic Activity From Monocytes/Macrophages
In concentrations up to 8 μg/mL, Ox-LDL stimulated monocyte/macrophage release of SMC mitogens in a dose-dependent manner (Fig 1A⇓). At higher concentrations of Ox-LDL, growth-promoting activity in CM decreased. CM from monocytes/macrophages incubated with native LDL did not stimulate SMC DNA synthesis. Addition of Ox-LDL without prior incubation with monocytes/macrophages was also ineffective on SMC DNA synthesis. CM from monocytes/macrophages incubated with Ox-LDL stimulated SMC proliferation, with maximal effect at an Ox-LDL concentration of 8 μg/mL (Fig 1B⇓).
Cytokine mRNA Production
mRNA levels for TNF-α IL-1β, HB-EGF, and PDGF-B were analyzed in monocytes/macrophages maintained in serum-containing medium for 12 hours and subsequently exposed to serum-free medium with or without 8 μg/mL native or Ox-LDL for another 6 hours. Initial cellular expression of TNF-α mRNA was low and remained so in both control medium and medium containing LDL. In monocytes/macrophages exposed to Ox-LDL for 6 hours, however, TNF-α mRNA expression was about twofold higher (Fig 2A and 2B⇓⇓). Control cells expressed high levels of IL-1β and moderate levels of HB-EGF mRNA but only low levels of PDGF-B mRNA. Expression of these mRNA species did not change in response to incubation with either native or Ox-LDL (Fig 3⇓). At 2 hours a decrease in mRNA levels was observed in all groups, an effect probably due to serum removal at the start of the experiment. After 6 hours all mRNA levels had returned to baseline levels.
Analysis of TNF-α and IL-1β Levels in Monocyte/Macrophage–CM
Determination of TNF-α levels in CM confirmed that LDL did not stimulate TNF-α secretion (Fig 4A⇓). Medium from cells incubated with 1 μg/mL Ox-LDL for 12 hours contained 323±1 versus 226±14 pg/mL pg/mL TNF-α in medium from control cells (P<.02). Maximal stimulation of TNF-α release (537±18 pg/mL, P<.005) was obtained at an Ox-LDL concentration of 8 μg/mL, whereas cell exposure to higher concentrations of Ox-LDL resulted in a reduction of TNF-α secretion. To determine whether the increased expression of IL-1β mRNA observed after a 6-hour incubation with Ox-LDL was associated with the activation of IL-1β release, IL-1β levels in CM were analyzed by ELISA. Addition of 8 μg/mL acetylated LDL increased TNF-α release (313±16 pg/mL) but was much less efficient than Ox-LDL, suggesting that the effect of Ox-LDL on TNF-α secretion was due to mechanisms other than scavenger receptor binding (data not shown). Incubation of cells with native LDL did not stimulate TNF-α release (Fig 4A⇓). A significant increase in IL-1β levels was seen in cell cultures exposed to 2 μg/mL LDL, but Ox-LDL and higher concentrations of native LDL had an inhibitory effect (Fig 4B⇓).
Effect of TNF-α on SMC DNA Synthesis
To determine the ability of TNF-α to induce DNA synthesis in human SMCs, growth-arrested cells were incubated with increasing concentrations of rTNF-α, which was found to stimulate [3H]thymidine uptake in a dose-dependent manner (Fig 5⇓). As described above, CM from monocytes/macrophages exposed to 8 μg/mL Ox-LDL contained ≈500 pg/mL TNF-α. This concentration of rTNF-α stimulated SMC DNA synthesis by twofold to threefold, ie, to about the same level of stimulation produced by CM from monocytes/macrophages exposed to 8 μg/mL Ox-LDL.
Blocking of TNF-α Activity in CM
To determine the relative importance of TNF-α in stimulating SMC DNA synthesis by monocytes/macrophages exposed to Ox-LDL, CM was preincubated for 1 hour with 0.05 μg/mL of a TNF-α–blocking antibody before the medium was added to the SMC cultures. Addition of this antibody reduced [3H]thymidine uptake by 44.7±8.4% (P<.005), whereas addition of an irrelevant antibody (mouse myeloma IgG) did not significantly influence [3H]thymidine uptake by SMCs (Fig 6⇓).
Transcription Factor Activity in Monocytes/Macrophages
The activity of redox-sensitive transcription factors NF-κB and AP-1 was analyzed in monocytes/macrophages exposed to control medium or medium containing 8 μg/mL native or Ox-LDL. High levels of active NF-κB were encountered in cells grown in control medium, and this level did not change in response to either native or Ox-LDL (Fig 7⇓). Control cells contained only minor amounts of active AP-1. A marked increase in the amount of active AP-1 occurred in cells exposed to Ox-LDL, whereas only a small increase was seen in cells incubated with native LDL. Reaction specificity was analyzed by adding excess unlabeled oligonucleotides, which resulted in the disappearance of the AP-1 band. Further evidence for reaction specificity was obtained by EMSA, which demonstrated that binding was eliminated by adding an antibody against c-Jun (Fig 8⇓). To exclude the possibility that AP-1 activation was induced by the TNF-α released from cells, monocytes/macrophages were incubated with Ox-LDL with or without a TNF-α–blocking antibody. This antibody did not affect AP-1 activation by Ox-LDL (Fig 9⇓, lanes 3 and 4) but did reduce AP-1 activation in response to rTNF-α (Fig 9⇓, lanes 5 and 6).
The mechanism whereby LDL is involved in the development of atherosclerosis remains to be fully understood. In animal models, increased LDL levels have been associated with the development of fatty streaks, composed mainly of lipid-filled macrophages.8 11 Increased LDL levels are also associated with a prolonged residence time for LDL in the arterial intima and consequently an increased risk of oxidation.6 7 Oxidation of LDL particles impairs their binding to LDL B,E receptors and thus confines their uptake mainly by macrophages via the scavenger receptor pathway.36
Because LDL oxidation is associated with the formation of a number of highly reactive substances, it is possible that these will activate macrophages to secrete cytokines and other growth factors. This hypothesis has been studied by several investigators, but the results of their analyses have not been consistent. Thus, Ox-LDL has been shown to induce synthesis of IL-8 and tissue factor in cultured monocytes but to suppress monocyte/macrophage expression of PDGF-B and TNF-α.23 24 37 These apparent inconsistencies may be partially due to differences in the techniques used for oxidative modification and in the concentrations of Ox-LDL used. The present results, as well as those of several other earlier studies, demonstrate that any stimulatory effects of Ox-LDL may be counteracted by inhibitory and cytotoxic factors at Ox-LDL concentrations of >10 to 15 μg/mL.15 38 This observation accords with that of the inhibitory effect on PDGF-B, TNF-α, and IL-1β expression of Ox-LDL concentrations >15 μg/mL.23 24 39
Studies from our group have demonstrated that Ox-LDL itself stimulates SMC growth but that this effect requires an additional mitogen, such as serum or PDGF.38 39B Hence, it is possible that the stimulatory effects of Ox-LDL observed in the present study are due to both an increased macrophage release of growth factors and potentiation of their mitogenic effect.
Monocyte/macrophage mRNA levels of PDGF-B and HB-EGF were not influenced by Ox-LDL. Nakano and coworkers40 have previously shown that lysoPC, which is formed from PC during LDL oxidation, increases macrophage expression of HB-EGF mRNA. The absence of a stimulatory effect of Ox-LDL on HB-EGF mRNA production in our experiments may have been due to insufficient levels of lysoPC (8 μg/mL Ox-LDL contains ≈2.0 μg/mL lysoPC) or at higher concentrations of Ox-LDL, to the presence of cytotoxic inhibitors such as oxysterols.
Exposure of monocytes to low concentrations of Ox-LDL increases mRNA expression and release of TNF-α and the release of SMC DNA synthesis–stimulating activity. At Ox-LDL concentrations >8 μg/mL, release of both TNF-α and mitogenic activity is inhibited. Addition of TNF-α–blocking antibodies to CM reduces growth-stimulatory activity by ≈50%. Taken together, these observations imply that the increased growth-stimulatory activity in CM from monocytes/macrophages exposed to Ox-LDL is due to activation of TNF-α synthesis and secretion.
The high levels of active transcription factor NF-κB and mRNA transcripts for IL-1β in our monocytes/macrophages under basal conditions indicate that these cells are in an activated state. Previous studies have shown that monocyte adhesion is associated with activation of IL-1β mRNA production.41 Hence, in the arterial intima, monocytes will release growth factors as a result of their adherence to extracellular matrix, and this release may be further enhanced if the monocytes are exposed to Ox-LDL trapped within the matrix. Such a process could play an important role in the transition of early fatty streaks into fibromuscular atherosclerotic lesions. The presence of Ox-LDL has been demonstrated in atherosclerotic plaques, but its actual concentration in the normal intima and atherosclerotic tissue remains unknown.42 However, it is likely that the concentration of Ox-LDL differs in different regions of the plaque, with low levels in the peripheral SMC-containing fibrous cap and higher levels in core regions. Accordingly, some regions of the plaque may contain levels of Ox-LDL that stimulate TNF-α release by monocytes/macrophages, whereas other regions may contain levels of Ox-LDL that either inhibit the secretory activity of these cells or are even cytotoxic or apoptotic.13 Macrophages from human atheromatous plaques have been shown to have higher levels of released TNF-α than do peripheral blood monocytes43 and TNF-α immunoreactivity has been demonstrated in human atherosclerotic plaques,44 but its relation to the presence of Ox-LDL remains to be determined.
The TNF-α promoter contains functional binding sites for transcription factors NF-κB and AP-1.45 Freshly isolated monocytes/macrophages contained high levels of active NF-κB but only minor amounts of AP-1. Cell exposure to 8 μg/mL Ox-LDL increased AP-1 levels but did not affect NF-κB activity. The functional importance of the AP-1 site in the TNF-α promoter is clearly demonstrated by the observation that a mutation in this site reduces both basal and phorbol 12-myristate 13-acetate–induced activity levels by 80% to 90%.46 Both NF-κB and AP-1 are activated by oxidizing agents such as H2O2.47 48 Because LDL oxidation is associated with the formation of a number of reactive oxygen species, it has been logical to assume that the biological effects of lipid oxidation are mediated by radical-dependent activation of these transcription factors. However, recent studies with cultured SMCs have indicated that a major signaling pathway involves activation of AP-1 by lysoPC.49
In conclusion, our results suggest that LDL oxidation is associated with the formation of factors that enhance macrophage release of TNF-α and that this process is mediated by activation of transcription factor AP-1. This mechanism may partially explain the association between hyperlipidemia and the intimal fibrosis that has been observed in atherosclerosis.
Selected Abbreviations and Acronyms
|DMEM||=||Dulbecco's modified Eagle's medium|
|ELISA||=||enzyme-linked immunosorbent assay|
|EMSA||=||electrophoretic mobility shift assay|
|HB-EGF||=||heparin-binding epidermal growth factor|
|Ox-LDL||=||oxidatively modified LDL|
|PCR||=||polymerase chain reaction|
|PDGF||=||platelet-derived growth factor|
|(r)TNF||=||(recombinant) tumor necrosis factor|
|SMC(s)||=||smooth muscle cell(s)|
This study was supported by grants from the Swedish Medical Research Council (No. 8311), the Swedish Heart and Lung Foundation, the Wallenberg Foundation, the Swedish Medical Society, King Gustaf V 80th Birthday Foundation, Prof Nana Svartz Foundation, Förenade Liv Mutual Group Life Insurance Co (Stockholm), Swedish Cancer Society, Nordic Academy for Advanced Study (No. 94.30.024/00), and the King Gustaf V and Queen Viktoria Foundation. FACS analyses were performed with the help of Ricardo Giscombe.
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