Hypoxia increases 25-hydroxycholesterol-induced interleukin-8 protein secretion in human macrophages

Atherosclerosis 170 (2003) 245–252

Hypoxia increases 25-hydroxycholesterol-induced interleukin-8 protein secretion in human macrophages Ellen Knutsen Rydberg∗ , Linda Salomonsson, Lillemor Mattsson Hultén, Kristina Norén, Göran Bondjers, Olov Wiklund, Tom Björnheden, Bertil G. Ohlsson Wallenberg Laboratory for Cardiovascular Research, Sahlgrenska University Hospital, Göteborg University, SE-41345 Göteborg, Sweden Received 1 July 2002; received in revised form 7 July 2003; accepted 14 July 2003

Abstract Interleukin-8 (IL-8) is a chemotactic factor for T-lymphocytes and smooth muscle cells and may therefore have an important effect in atherogenesis. It is secreted from oxysterol-containing foam cells which have been found in hypoxic zones in atherosclerotic plaques. The aim of this study was to investigate the effect of hypoxia on the secretion of IL-8 by oxysterol-stimulated macrophages. Hypoxia enhances 25-hydroxycholesterol (25-OH-chol)-induced IL-8 secretion in human monocyte-derived macrophages. The effect is most pronounced when macrophages are incubated with low concentrations of 25-OH-chol. Both 25-OH-chol and hypoxia increases the intracellular level of the signalling molecule hydrogen peroxide (H2 O2 ). This event coincided with an enhanced binding of the transcription factor c-jun to the IL-8 gene promoter and an increased IL-8 mRNA expression in hypoxic macrophages. These observations suggest that similar intracellular signalling pathways are used for both 25-OH-chol-induced IL-8 expression and hypoxia-induced IL-8 expression. Thus, hypoxia in atherosclerotic plaques may increase the secretion of IL-8 from oxysterol-containing foam cells, which subsequently may accelerate the progression of atherosclerosis. © 2003 Published by Elsevier Ireland Ltd. Keywords: Atherosclerosis; c-jun; Hydrogen peroxide; 25-Hydroxycholesterol; Hypoxia; Interleukin-8; Macrophage

1. Introduction The atherosclerotic lesion is characterized by an enrichment of macrophages and T-lymphocytes and an increased number of smooth muscle cells in the vessel wall. Migration and proliferation of smooth muscle cells are considered to be a major contributor to atherosclerotic plaque formation [1]. Interleukin-8 (IL-8) is mitogenic and chemotactic for smooth muscle cells and T-lymphocytes [2]. Furthermore, IL-8 induces endothelial cell proliferation [3] and is an important mediator of angiogenesis [4]. LDL receptor-deficient mice with bone marrow cells lacking CXCR2, one of the high-affinity receptors for IL-8, have significantly less atherosclerosis than mice with wild-type bone marrow cells [5]. Together these data suggest that IL-8 makes multiple contributions to atherosclerosis development. Oxidized low-density lipoprotein (oxLDL) and components in oxLDL such as oxysterols accumulate in lipid-filled ∗

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0021-9150/$ – see front matter © 2003 Published by Elsevier Ireland Ltd. doi:10.1016/S0021-9150(03)00302-2

macrophages in atherosclerostic plaques [6]. An increased IL-8 secretion is found in these ‘foam cells’ [7,8] and enlarged IL-8 mRNA levels are found in atherosclerotic lesions from atherectomized human carotid arteries as well as in macrophage foam cells in human atheroma [4]. We have previously shown that foam cells derived from atherosclerostic plaques contain oxysterols [9] and that oxysterols in oxLDL increase IL-8 secretion in macrophages [7]. In contrast to other oxysterols, 25-hydroxycholesterol (25-OH-chol) regulates the expression of a wide variety of proteins already at low concentrations. Thus, 25-OH-chol increases the secretion of IL-1␤ protein in monocytes [10] as well as IL-8 in macrophages [7]. 25-Hydroxycholesterol also decreases sterol synthesis and the expression of cellular LDL receptors [11], and the lipoprotein lipase expression [9]. Intracellular 25-OH-chol is bound to the oxysterol binding protein (OSBP), which has high affinity for this oxysterol in comparison to other oxysterols [12]. During atherogenesis, the thickness of the arterial wall increases, leading to an impaired diffusion, which results in an oxygen (O2 ) and nutrient deficiency in the more deeply

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situated parts of the arterial wall. At the same time, O2 consumption is augmented during atherogenesis [13], which may result in an energy imbalance in the arterial wall, due to an increased number of foam cells. In vivo studies show that zones of hypoxia occur within atherosclerotic plaques [14] and local hypoxia may play a key role in atherogenesis [15]. Hypoxia inhibits macrophage migration, which leads to macrophage accumulation in hypoxic areas [16]. In healthy tissues, the O2 tension is generally between 20 and 70 mmHg (i.e. 2.5–9.0% O2 ), whereas inadequate perfusion of diseased tissues can cause areas of hypoxia in which O2 tensions of <10 mmHg (i.e. <1% O2 ) have been reported [17]. The molecular mechanisms by which mammalian cells sense hypoxia and transduce this signal have not been resolved [18]. Recent studies show that the respiratory chain plays an important role in O2 sensing [19]. Hypoxia activates macrophages leading to formation of mitochondria reactive oxygen species (ROS), which act as inducers of hypoxic genes [20]. In vascular diseases, oxidative stress regulates a variety of redox-sensitive genes and signalling pathways [21] and endothelial cells exposed to hypoxia increase IL-8 expression [22]. The effect of hypoxia on atherosclerotic plaque development has not been studied in detail. The aim of this study was to investigate whether hypoxia could further increase IL-8 secretion by macrophages incubated with oxysterols. The results from this study show that hypoxia enhances the 25-OH-chol-induced IL-8 secretion in macrophages, probably mediated via elevated intracellular H2 O2 levels and activator protein-1 (AP-1) binding to the IL-8 promoter. Therefore, hypoxic areas in atherosclerotic plaques, which co-localise with oxysterol-containing macrophages, may contribute to an enhanced development of the atherosclerotic lesion by promoting the release of IL-8 secretion.

2. Materials and methods 2.1. Preparations of macrophages Human mononuclear cells were isolated by Ficoll-Paque centrifugation (Pharmacia, Uppsala, Sweden) in a discontinuous gradient from buffy coats obtained from the blood bank at Sahlgrenska University Hospital, Göteborg, Sweden as previously described [23]. Cells were seeded in serum-free medium, macrophage-SFM (GIBCO/BRL, Grand Island, NY, USA), supplemented with 100 U/ml penicillin and 100 ␮g/ml streptomycin. Cells were differentiated in macrophage-SFM medium with antibiotics supplemented with 70 U/ml human granulocyte macrophage colony-stimulating factor (GM-CSF) (R&D Systems Europe Ltd., Oxon, UK). After 3 days, the medium was changed to macrophage-SFM without GM-CSF and cells were cultured up to 8 days. When analyzed by FACS, 95% of the monocyte-differentiated macrophages expressed the macrophage marker CD 68 (results not shown).

2.2. Cell experiments 2.2.1. Hypoxia experiments The experiments were started 8 days after the cells were plated. The medium was pre-equilibrated at the desired O2 concentration, before it was added to the cells. In some experiments macrophages were incubated in a humid chamber at 37 ◦ C for 24 h with a constant gas flow of different O2 concentrations (21, 3, 2, 1 and 0%) at 5% CO2 and with the balance made up of N2 . When mRNA expression was investigated, macrophages were incubated for 0, 6, 12, and 24 h at normoxia (21% O2 ) and hypoxia (0% O2 ) at 5% CO2 with 74% N2 (at normoxia) and 95% N2 (at hypoxia). After incubation, either the cell media and the total cell protein or total RNA was collected. Hypoxia-treated cells were harvested in a chamber with a constant gas flow of 100% N2 and cells incubated at normoxia were harvested under normal O2 conditions. 2.2.2. Experiments with oxysterols and/or hypoxia Macrophages were incubated with oxysterols at hypoxia 8 days after cell preparation. During the first 24 h of the experiments, the macrophages were incubated with oxysterols at normoxia, to allow them to fill with oxysterols before exposing the cells to hypoxia. This incubation was followed by another incubation for 24 h with oxysterols, in cell media equilibrated at either normoxia or hypoxia as described above. The oxysterols used were 25-OH-chol (Sigma, St. Louis, MO, USA) at 1.0, 3.0, 4.0 and 5.0 ␮g/ml, 27-hydroxycholesterol (27-OH-chol) (Research Plus Inc., Bayonne, NJ, USA) at 0.5, 1.0, 3.0, 5.0 and 10.0 ␮g/ml or 7-ketocholesterol (7-keto-chol) (Sigma) at 5.0 ␮g/ml. The oxysterols were dissolved in 95% ethanol and added to the medium at a ratio of 1 ␮l/ml of medium for 25-OH-chol and 7-keto-chol and 2 ␮l/ml medium for 27-OH-chol. To normoxia-incubated control cells, only the ethanol vehicle was added. Nuclear protein extracts (NEs), total cell protein, and the media were collected. In some experiments, differentiated macrophages were incubated at either normoxia or hypoxia, in the presence or absence of 10 ␮M vitamin E, ␣-tocopherol (Sigma). 2.2.3. Chemical hypoxia experiments Macrophages were incubated at normoxia with 1.25, 2.5, 5.0 or 10.0 ␮g/ml oligomycin (Sigma) for 24 h. In one experiment macrophages were incubated at hypoxia for comparison. Oligomycin was dissolved in 95% ethanol and added to the medium at a ratio of 1 ␮l/ml medium. To normoxia-incubated control cells, only the ethanol vehicle was added. In one experiment 10 ␮M vitamin E (Sigma), was added to the medium containing 5 ␮g/ml oligomycin. Media and total cell protein were collected for analysis. 2.3. Cell viability Potential cytotoxic effects of the culture conditions were measured as lactate dehydrogenase (LDH) leakage,

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using a kit purchased from Roche Diagnostics (Mannheim, Germany). LDH leakage was below 15% in all samples, confirming that cells were viable under all culture condition. Similar results were also seen with trypan blue exclusion and the pH of the media did not change during the experiments. 2.4. Interleukin-8 protein secretion and mRNA expression The IL-8 protein secretion in the cell culture media was determined in relation to total cell protein using Quantikine enzyme-linked immunosorbent assay (ELISA) kit from R&D Systems. Total RNA was isolated using an RNeasy Mini Kit (Qiagen GmbH, Hilden, Germany). Real-time reverse transcriptase polymerase chain reaction (RT-PCR) was performed by the TaqMan pre-Developed IL-8 RT-PCR kit (Applied Biosystems, Foster City, CA, USA). The RT reaction was performed on a Gene Amp PCR system 9700 (Applied Biosystems) and the PCR amplification was performed on an ABI PRISM 7700 sequence detection system (Applied Biosystems). Actin mRNA was used as house-keeping gene and PCR amplification was performed for 40 cycles. 2.5. Measurements of hydrogen peroxide Intracellular levels of hydrogen peroxide (H2 O2 ) were determined in macrophages incubated at either normoxia or hypoxia with and without 1 or 5 ␮g/ml 25-OH-chol or 5 ␮g/ml 7-keto-chol. Macrophages were also incubated with 2.5 or 5 ␮g/ml oligomycin at normoxia. After 24 h of incubation, cells were harvested in 0.5 ml distilled water and kept at −80 ◦ C for 1.5 h before they were thawed and analyzed. All cells were treated in a similar way. No degradation of H2 O2 in the cell culture medium was found after 2 h, when H2 O2 was added to the medium at +4 ◦ C. The levels of H2 O2 in the cell lysates were analyzed by a specific high-sensitivity Bioxytech H2 O2 -560 assay (OXIS International Inc., Portland, OR, USA) based on the oxidation of ferrous ions (Fe2+ ) to ferric ions (Fe3+ ) by H2 O2 at acidic pH. The ferric ion binds to the indicator dye (xylenol-orange), to form a stable colored complex, measured at 560 nm. Once formed, the complex is stable for up to 12 h. Hydrogen peroxide levels were expressed in relation to total cellular protein. 2.6. Preparation of nuclear protein extracts Nuclear protein extracts were prepared as described [23]. Extracts from macrophages incubated at 0% O2 were prepared in 100% N2 , while extracts from cells incubated at 21% O2 were prepared under normal O2 conditions. Recombinant human c-jun, which forms the homodimer complex of the activator protein-1 complex, was purchased from Promega Corp., Madison, WI, USA.

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2.7. Electrophoretic mobility shift assay An oligonucleotide with a binding site for AP-1, corresponding to the region −146 to −103 on the IL-8 promoter was used to detect binding of AP-1 and an oligonucleotide containing the consensus binding site for CREB was purchased from Santa Cruz Biotechnology, Santa Cruz, CA, USA. Oligonucleotides (Life Technologies AB, Täby, Sweden) were annealed and purified as described [23]. A double-stranded oligonucleotide with the sequence 5 -GCCCTACGTGCTGTCTCA-3 [24] was used to detect binding of HIF-1. These oligonucleotides were used for electrophoretical mobility shift assay (EMSA) as earlier described [23] and an equal protein amount of NE from macrophages incubated at either normoxia or hypoxia with and without 5 ␮g/ml 25-OH-chol was used. In some experiments, rabbit polyclonal immunoglobulin G (IgG) towards c-jun and c-fos (Santa Cruz Biotechnology) was added to the reactions to identify binding of these transcription factors to the oligonucleotide. 2.8. Protein estimation Total cell protein was harvested in 0.2 M NaOH. The protein concentrations for both total cell protein and NEs were determined according to Bradford. 2.9. Data analysis Data are presented as means ± standard deviation (S.D.). Student’s two-tailed paired t-test was used and complemented with the non-parametric test, Wilcoxon signed-rank sum test. A P-value of <0.05 was considered statistically significant. 3. Results 3.1. Interleukin-8 protein secretion and mRNA expression in hypoxia-treated macrophages The IL-8 protein secretion in the normoxia-incubated control cells varied from 0.45 to 5.18 pg/␮g cell protein between the donors used. A significant increase of the IL-8 protein secretion was found when macrophages were incubated for 24 h at decreasing O2 concentrations, in comparison to macrophages incubated at normoxia (Fig. 1). The IL-8 mRNA expression in macrophages incubated at hypoxia was significantly higher ( P < 0.05) after 12 h of incubation compared to cells incubated at normoxia at the same time (Fig. 2). 3.2. Effects of vitamin E on hypoxia-induced interleukin-8 protein secretion Macrophages were incubated at hypoxia, in the presence or absence of vitamin E, a scavenger of free radicals, to

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IL-8 secretion (fold increase over normoxia)

3.5

Table 1 Effects of Vitamin E on hypoxia-induced IL-8 protein secretion

***

3

Treatment

*

Fold increase over normoxia

2.5

Normoxia Normoxia + Vitamin E (10 ␮M) Hypoxia Hypoxia + Vitamin E (10 ␮M)

2

* 1.5 1 0.5

n=6

n=6 n=6

21%

3%

0 2%

1%

1 0.82 2.48 1.56

N/A 0.42 0.92 0.78

# ∗

0%

Oxygen level Fig. 1. Effects of hypoxia on the IL-8 protein secretion in human macrophages. The IL-8 protein secretion in cell culture medium from macrophages incubated for 24 h in 3, 2, 1 and 0% O2 gas mixture. N denotes the number of donors. Due to the numbers of incubators available for each oxygen concentration the incubations of the macrophages from various donors were performed like this: macrophages from six donors were incubated at 3, 0 and 21% O2 and macrophages from six other donors were incubated at 2, 0 and 21% O2 . Furthermore, six other donors were incubated at 1, 0, and 21% O2 and macrophages from two donors were incubated at 0 and 21% O2 . Protein secretion at normoxia was set to 1. The IL-8 protein secretion in control cells varied from 0.45 to 5.18 pg/␮g cell protein. Values are mean ± S.D. The IL-8 secretion at normoxia was compared to the secretion in macrophages incubated at lower oxygen concentrations. P < 0.05; P < 0.001 by Wilcoxon signed-rank sum test.

investigate whether the increased IL-8 secretion could be mediated by ROS. The results confirm that there is a significant increase in IL-8 protein secretion when macrophages are incubated at hypoxia, while vitamin E significantly IL-8 mRNA /Actin mRNA (fold increase over normoxia, at 0h)

S.D.

Macrophages incubated at either normoxia or at hypoxia in the presence or absence of 10 ␮M Vitamin E, for 24 h. The IL-8 protein secretion at normoxia without Vitamin E was set to 1. Values are mean±S.D. (n = 6). ∗ P < 0.05 vs. hypoxia. # P < 0.05 vs. normoxia.

n=20

n=20

Mean

Student’s t-test

(∗ P < 0.05) decreases this hypoxia-induced IL-8 secretion (Table 1). These observations suggest that ROS may be involved in hypoxia-induced IL-8 secretion. 3.3. Effects of oligomycin on interleukin-8 secretion To mimic hypoxic conditions with an insufficient respiratory chain reaction, adenosine triphosphate (ATP) synthesis, and a possible ROS formation, macrophages were incubated at normoxia by increasing concentrations of oligomycin. Oligomycin blocks F0 F1 ATP synthase, a protein complex, using energy developed under the respiratory chain reaction to drive ATP synthesis [25]. For comparison, macrophages were also treated with hypoxia. A significant increase in IL-8 protein secretion was found when increasing concentrations of oligomycin were used (Fig. 3). The oligomycin-induced IL-8 secretion was more pronounced than the hypoxia-induced IL-8 secretion. Vitamin E decreased IL-8 secretion in macrophages incubated with oligomycin (Fig. 3). These observations show that

**

5 4.5 4 3.5 3 2.5 2 1.5 1

Hypoxia

0.5

Normoxia

0 0

4

8

12

16

20

24

Time (h) Fig. 2. Effects of hypoxia on the IL-8 mRNA expression. Macrophages incubated at normoxia and hypoxia for 0, 6, 12 and 24 h. The IL-8 mRNA expression at normoxia at the beginning of the experiment (0 h) was set to 1 and actin was used as house-keeping gene. Values are mean ± S.D. (n = 3). P < 0.01 by Student’s two-tailed paired t-test.

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Fig. 3. Effects of oligomycin on the IL-8 secretion. Macrophages incubated at either hypoxia or at normoxia in the presence or absence of oligomycin (␮g/ml). In one experiment 10 ␮M Vitamin E, was added together with 5 ␮g/ml oligomycin. Interleukin-8 protein secretion at normoxia was set to 1. Values are mean ± S.D. (n = 3–6). P < 0.05; P < 0.01 by Student’s two-tailed paired t-test.

exposure of cells to 5 ␮g/ml 25-OH-chol together with hypoxia. However, hypoxia had its most pronounced effect on the 25-OH-chol-induced IL-8 secretion when low concentrations of 25-OH-chol (1 and 3 ␮g/ml) were used.

oligomycin increases IL-8 secretion and that ROS might be involved in this elevated IL-8 secretion. 3.4. Interleukin-8 protein secretion in macrophages treated with 25-hydroxycholesterol and hypoxia

3.5. Effects of oxysterols and hypoxia on the intracellular level of hydrogen peroxide

An increasing IL-8 protein secretion is found when macrophages are incubated at normoxia with increasing concentrations of 25-OH-chol (Fig. 4), but not with 7-keto-chol or 27-OH-chol (results not shown). Interestingly, hypoxia increases this 25-OH-chol-induced IL-8 secretion. The highest level of IL-8 protein secretion was found after

The intracellular molecule H2 O2 is considered to belong to the group of ROS, which activate various intracellular signalling pathways in the cell [26]. We investigated whether hypoxia or 25-OH-chol could increase the intracellular level

14 12 10

** 8

**

6 4

***

2

Hypoxia Normoxia

0 0

1

3

4

5

Fig. 4. Effects of 25-OH-chol on the hypoxia-induced IL-8 protein secretion. Macrophages incubated for 24 h with increasing concentrations of 25-OH-chol, followed by another incubation for 24 h with 25-OH-chol, in cell media equilibrated at normoxia or at hypoxia. Values are mean ± S.D. (n = 6). The IL-8 secretion at normoxia was compared with IL-8 secretion at hypoxia for each concentration of 25-OH-chol used. P < 0.01; P < 0.001 by Wilcoxon signed-rank sum test.

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Table 2 Effects of hypoxia and 25-OH-chol on the intracellular level of H2 O2 in macrophages Treatment

Normoxia Normoxia + 25-OH-chol (1 ␮g/ml) Normoxia + 25-OH-chol (5 ␮g/ml) Hypoxia Hypoxia + 25-OH-chol (1 ␮g/ml) Hypoxia + 25-OH-chol (5 ␮g/ml)

nmol H2 O2 /mg cell protein Mean

S.D.

0.29 0.32 1.83 0.56 0.48 1.92

0.18 0.17 0.64 0.20 0.20 0.95

Student’s t-test

∗ ∗ ∗,## ∗

The intracellular level of H2 O2 after 24 h of incubation at normoxia or at hypoxia with or without 25-OH-chol (1 and 5 ␮g/ml). Values are mean ± S.D. (n = 4). ∗ P < 0.05 vs. normoxia. ## P < 0.01 vs. normoxia + 25-OH-chol (1 ␮g/ml).

of H2 O2 in macrophages, which may subsequently contribute to the increased IL-8 secretion found in these cells. Macrophages incubated either at hypoxia or with 5 ␮g/ml 25-OH-chol significantly increased the intracellular level of H2 O2 (Table 2), although hypoxia had no further effect on 25-OH-chol-induced H2 O2 levels, when high 25-OH-chol concentration (5 ␮g/ml) was used. Instead hypoxia significantly (## P < 0.01) enhanced a 25-OH-chol-induced H2 O2 level when 1 ␮g/ml 25-OH-chol was used. In contrast, H2 O2 levels were not affected by 7-ketochol (results not shown). The intracellular level of H2 O2 tended to increase in macrophages incubated with oligomycin. However, these results were not statistically significant (results not shown). 3.6. Effects of hypoxia and 25-hydroxycholesterol on the binding of the transcription factor activator protein-1 to the interleukin-8 promoter Binding of an AP-1 complex [27] to a regulatory element on the IL-8 promoter was investigated in macrophages incubated at normoxia or at hypoxia in the presence or absence of 25-OH-chol-. Both 25-OH-chol and hypoxia-induced binding of the AP-1 complex to the IL-8 promoter, as showed with electrophoretic mobility shift assay (EMSA) (Fig. 5). Similar results were obtained when NEs from four donors were used. The transcription factor complex AP-1 is formed either as a homodimer of c-jun or as a heterodimer of c-jun and c-fos. Specific antibodies against c-jun and c-fos were used to identify the constituents of this AP-1 complex. Antibodies against c-jun, but not against c-fos, supershifted the AP-1 complex (Fig. 5). These results suggest that the AP-1 complex, bound to this site on the IL-8 promoter in macrophages, contains the transcription factor c-jun. This observation was further confirmed when recombinant c-jun was found to bind to this site on the IL-8 promoter (Fig. 5). Hypoxia further increased binding of the transcription factors HIF-1 and CREB to their specific binding sites (results not shown), although no binding sites for these transcription

Fig. 5. Effects of 25-OH-chol and hypoxia on the binding of AP-1 to the IL-8 promoter in macrophages. Nuclear protein extracts from macrophages incubated at normoxia or at hypoxia, in the presence or absence of 5 ␮g/ml 25-OH-chol. Protein binding to a ␣32 P-labeled DNA oligonucleotide corresponding to the position −146 to −103 in the IL-8 promoter was analyzed by EMSA. Specific antibodies against the transcription factors c-jun and c-fos were added to some of the binding reactions. An antibody against c-jun was also added to a binding reaction containing only the DNA probe and recombinant c-jun was included in one binding reaction.

factors were found within the first 2.3 kB on the human IL-8 promoter.

4. Discussion In this study, we found that hypoxia significantly increase a 25-OH-chol-induced IL-8 protein secretion in human macrophages, when low concentrations of 25-OH-chol are used. Oxysterols, particular 25-OH-chol, increases IL-8 [7] and IL-1␤ [10] secretion from macrophages. We have previously shown that macrophages derived from atherosclerotic plaques contain 25-OH-chol [9] and that macrophages are found within hypoxic areas in atherosclerotic plaques [14]. This suggests that an increased IL-8 protein secretion may be released from hypoxic macrophages in atherosclerotic lesions. Increased IL-8 protein secretion could enhance the development of the atherosclerotic plaque, since IL-8 recruits smooth muscle cells [1], T-lymphocytes [2], and induces endothelial cell proliferation [3]. In this study, we also found that hypoxia by itself increases the constitutive IL-8 secretion by human macrophages. Similar results were also reported by Hirani et al. who showed an up-regulation

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of IL-8 protein and mRNA expression in hypoxic human macrophages and an increase of IL-8 expression in rabbit alveolar macrophage after acute hypoxia treatment [28]. Our results suggest that macrophages exposed to only hypoxia increase the IL-8 secretion to a certain extent, which is further increased when macrophages are incubated together with lower concentrations of 25-OH-chol. Signalling by intracellular H2 O2 appears to participate in the elevated IL-8 secretion, since increased intracellular levels of H2 O2 were found in macrophages incubated at hypoxia. Hydrogen peroxide is generated when hypoxia inhibits the activity of cytochrome oxidase in the respiratory chain [18]. A putative role for H2 O2 in the secretion of IL-8 was further confirmed when a decreased secretion of IL-8 was found when hypoxic macrophages were incubated with vitamin E, a scavenger of free radicals. The decreased effect on IL-8 secretion caused by vitamin E was also found when macrophages were incubated with vitamin E together with oligomycin, an inducer of chemical hypoxia. Increased IL-8 secretion induced by 5 ␮g/ml 25-OH-chol, coincided with an enhanced intracellular level of H2 O2 , suggesting that this secretion is also mediated by H2 O2 . At high concentrations of 25-OH-chol (5 ␮g/ml), no additional increase of the intracellular H2 O2 level was found when these cells were incubated at hypoxia. This observation may explain why IL-8 secretion is not further increased by hypoxia when macrophages are incubated with high concentrations of 25-OH-chol. Instead hypoxia treatment significantly increases both the 25-OH-chol-induced IL-8 secretion as well as the 25-OH-chol-induced intracellular H2 O2 level, when cells were incubated with low concentration of 25-OH-chol. These observations suggest that both hypoxia and 25-OH-chol activate intracellular pathways which result in an increased formation of intracellular H2 O2 and that H2 O2 is one of the factors that contribute to an increased IL-8 secretion. The increased binding of a c-jun-containing AP-1 complex to the IL-8 promoter in macrophages incubated with 25-OH-chol or at hypoxia, suggests that this transcription factor complex is involved in the increased IL-8 secretion found in these cells. The AP-1 complex is known to be activated by free radicals [29] and in cells incubated at hypoxia [30]. Hydrogen peroxide is an important intracellular molecule that participates in the activation of the transcription factor c-jun found in the AP-1 complex. In this study, we found that both 25-OH-chol and hypoxia increase the intracellular level of H2 O2 which may be one of the factors involved in the enhanced binding of the transcription factor c-jun to the IL-8 promoter and subsequently increase IL-8 expression. It is likely that 25-OH-chol and hypoxia, which influence the same intracellular targets, have an additive effect on the IL-8 secretion. This additive effect could also explain why the IL-8 secretion only reaches a certain level irrespective of increased 25-OH-chol concentration. In conclusion, results from this study suggest that hypoxia increases 25-OH-chol-induced IL-8 secretion from

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macrophages, probably mediated via increased intracellular H2 O2 levels and AP-1 binding to the IL-8 promoter. Therefore, hypoxic areas in atherosclerotic plaques, which co-localise with oxysterol-containing macrophages, may contribute to an enhanced development of the atherosclerotic lesion by promoting the release of IL-8 secretion.

Acknowledgements This work was supported by grants from the Swedish Medical Research Council (project no. 4531), Sahlgrenska University Hospital funds for clinical research and the Swedish Heart and Lung Foundation.

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