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【摘要】
Objective- Modified lipoproteins induce inflammatory reactions in the atherosclerotic arterial wall. We have previously found that macrophages in atherosclerotic lesions secrete lysosomal hydrolases that can modify low-density-lipoprotein (LDL) in vitro to generate "hydrolase-modified LDL" (H-LDL). Here, we studied whether H-LDL exerts inflammatory effects on cultured human macrophages.
Methods and Results- Using cytokine cDNA arrays, we found that H-LDL induced expression of IL-8, but not of the anti-inflammatory cytokines IL-10 and transforming growth factor (TGF)-ß, in human monocyte-derived macrophages. H-LDL induced rapid phosphorylation of the p38 mitogen-activated protein kinase (MAPK), nuclear translocation of 2 transcription factors, nuclear factor B (NF- B) and activator protein 1 (AP-1), and time-dependent secretion of IL-8 from the macrophages. Inhibition of MAPKs and of transcription factors showed that p38 MAPK and NF- B, but not ERK1/2, JNK, or AP-1, were crucial for the H-LDL-induced IL-8 secretion from the macrophages.
Conclusions- The results show that by activating p38 MAPK and NF- B, macrophage hydrolases modify LDL into biologically active particles capable of triggering the secretion of IL-8 in macrophages. Thus, activated hydrolase-secreting macrophages in atherosclerotic lesions may sustain a proatherogenic extracellular environment by hydrolyzing LDL and triggering it to act in an autocrine or paracrine fashion to induce IL-8 secretion by the plaque macrophages.
LDL modified by secreted macrophage-derived lysosomal hydrolases (H-LDL) induce secretion of IL-8 by cultured human monocyte-derived macrophages. Secretion of IL-8 by H-LDL-stimulated macrophages depended on the activation of p38 MAPK and NF- B transcription factor.
【关键词】 atherosclerosis cytokines inflammation LDL
Introduction
Atherogenesis is characterized by migration of monocytes into the arterial intima and their transformation into macrophage-derived foam cells. IL-8, a chemotactic cytokine, has been recently shown to induce firm adhesion of monocytes to endothelial cells under flow conditions in vitro 1 to be present in human atherosclerotic lesions 2 and to have a causal role in the development of experimental atherosclerosis. 3
Although low-density lipoprotein (LDL) modifications are thought to be a prerequisite for atherosclerosis, the actual types of modifications that drive atherogenesis in the human arterial intima are currently not known. LDL particles undergo oxidative modification to some extent in arterial wall, 4 and oxidized LDL induces expression of IL-8 in monocytes 5 and macrophages 2 in vitro. In addition, there is evidence for enzymatic modification of LDL in the arterial intima, 6 leading to the generation of morphologically modified unesterified cholesterol-rich particles with proinflammatory properties, such as secretion of IL-8 from endothelial cells. 7 Isolated LDL particles treated with trypsin and cholesteryl esterase, ie, enzymatically-modified LDL (E-LDL) have served as an in vitro model for this type of modification. Stimulated by these findings, we recently hypothesized that lysosomal proteases and lipases derived from arterial cells would modify LDL in atherosclerotic lesions. We have provided support for this hypothesis by showing the presence of cathepsin D and lysosomal acid lipase extracellularly in human atherosclerotic lesions. 8 Moreover, in the presence of inflammatory stimuli, cultured macrophages can release hydrolases that generate morphologically modified LDL, hydrolase-modified LDL (H-LDL), capable of generating foam cells from macrophages and smooth muscle cells, when added to these cells in vitro. 8
In this study we elucidated the effects of H-LDL on the expression of IL-8 in cultured human macrophages and also partially characterized the intracellular signaling induced by H-LDL in macrophages. We found that H-LDL triggered expression of IL-8 in macrophages, and that the secretion of IL-8 involved activation of the p38 mitogen-activated protein kinase (MAPK) and nuclear factor B (NF- B).
Methods
Preparation and Labeling of LDL
Human LDL (d=1.019 to 1.050 g/mL) was isolated and labeled as described previously. 8
Preparation of H-LDL
H-LDL was prepared as described. 8 Briefly, LDL was incubated for 18 hours with macrophage-preconditioned medium, ie, the medium in which the opsonized zymosan-treated macrophages had been incubated for 24 hours. After the incubation, modified LDL was purified by ultracentrifugation and incubated for 30 minutes with 10 µg/mL of polymyxin (Sigma) at 37°C to remove endotoxins. The endotoxin content of 50 µg/mL of H-LDL, which was the concentration of H-LDL to which macrophages were exposed, was measured by the Limulus Amebocyte Lysate kit (Cambrex) and was found to be 0.47±0.19 EU/mL, ie, below the concentration of 1.3±0.2 EU/mL (or 100 pg/mL of lipopolysaccharide ), which we found to be without any detectable effect on the secretion of IL-8 from macrophages. The amount of H-LDL is expressed in terms of its total cholesterol content, which was determined by the CHOD-PAP-kit (Roche); 50 µg total cholesterol/mL of H-LDL equals to 25 µg protein/mL.
Cell Culture
Human monocytes were isolated and differentiated into macrophages as described. 8 Before the experiments, growth of the macrophages was arrested by incubating them for 3 to 18 hours in the absence of growth factors.
Analysis of the Cytokine Expression and Secretion in Cultured Vascular Cells
The cells were incubated with H-LDL (50 µg total cholesterol/mL) for the times indicated and total RNA was extracted with Qiagen Mini Kit, and stored at -70°C, if not used immediately. Expression of 96 common human cytokine genes in cultured cells was screened with the Human Common Cytokine Gene Array (Superarray) kit according to the manufacturer?s instructions. For reverse-transcription polymerase chain reaction (RT-PCR), 1 µg of total RNA was reverse-transcribed to cDNA using Superscript pre-amplification system, random primers, and MMLV Reverse Transcriptase (Invitrogen). The primers for amplification of IL-8 by PCR were as follows: IL-8: 5'-ATGACTTCCAAGCTGGCCGTGGC-3' (S), 5'-CTCAGCCCTCTTCAAAAACTTCTCC-3' (AS); glyceraldehyde-3-phosphate dehydrogenase (GAPDH): 5'-ACCACAGTCCATGCCATCAC-3' (s), 5'-TCCACCACCCTGTTGCTGTA-3' (AS). The products were analyzed in agarose gel (1.4%) containing ethidium bromide and the bands were quantified by the Gel Doc 2000 gel documentation system. The amount of IL-8 in the conditioned media was determined by commercial enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems).
Analysis of the MAPKs
Macrophages were preincubated with inhibitors of the MAPKs, ie, against p38 (SB203580, 10 µmol/L), extracellular-signal regulated kinases 1 and 2 (ERK1/2) (PD98059, 30 µmol/L), and c-Jun-N-terminal kinase (JNK) (JNK inhibitor II, 5 µmol/L). H-LDL was then added and incubations continued for 6 hours at 37°C. After the incubation, secretion of IL-8 into the cell culture media was determined by ELISA. H-LDL and inhibitors were not cytotoxic toward the cells as measured by the release of lactate dehydrogenase from the cells with Cytotoxicity Detection Kit (Roche). For Western blots of p38, macrophages were stimulated for the indicated times with H-LDL, and cellular proteins (50 µg) were run on 15% SDS-PAGE. The proteins were transferred into polyvinylidene fluoride (PVDF) membranes at 4°C, and the bands were detected either by rabbit polyclonal anti-phospho-p38 (#9211, 1:1000; Cell Signaling Technology) or antibody against p38 protein (#9212, 1:1000; Cell Signaling Technology), and with horseradish peroxidase-conjugated secondary antibody (#7074, 1:2000; Cell Signaling Technology). The immunoblot signals were visualized with enhanced chemiluminescence kit (GE Healthcare).
Analysis of the Transcription Factors
Nuclear proteins from H-LDL-stimulated and control macrophages were extracted as described previously. 9 Electrophoretic mobility shift assay was then performed as described previously. 10 Briefly, nuclear protein extracts (4 to 10 µg) were incubated for 60 minutes with a [ - 32 P]ATP-labeled specific DNA probe (50 000 dpm) and run on 4% nondenaturing PAGE gel. In some experiments, unlabeled competitor probes were added 10 minutes before the labeled probes. The gel was then dried and the bands were visualized by autoradiography. In supershift assays, 2 µg of affinity-purified polyclonal antibodies were added after the binding reactions and incubation was continued for 1 hour at RT.
Macrophages were preincubated with inhibitors of NF- B, ie, CAPE (15 µg/mL) and sulfasalazine (2 mmol/L), or with an inhibitor of AP-1 (curcumin, 10 µmol/L). H-LDL was then added and incubations continued for 6 hours at 37°C. After the incubation, media were centrifuged at 4°C for 5 minutes at 400 g, and IL-8 in the media was determined by ELISA.
Other Assays
The degree of oxidation of H-LDL was determined by measuring the amounts of thiobarbituric acid-reactive substances (TBARS). 11 The protein concentration of the SDS-PAGE samples was determined with RC DC Protein Assay Kit (Bio Rad).
Statistical Analysis
The data shown in all the figures are expressed as means of the indicated numbers of experiments ±SEM. The Student t test was used for evaluation of the significance of difference between experimental groups and P <0.05 were considered statistically significant and probability values under 0.01 highly significant.
Results
First, the expression of inflammatory cytokine genes in H-LDL-treated human monocyte-derived macrophages was screened with a cDNA array ( Figure 1 ). For the experiments, H-LDL was first prepared by incubating LDL with conditioned media from opsonized zymosan-treated macrophages, resulting in hydrolysis of LDL apoB-100 and lipids, as described previously. 8 Butylated hydroxytoluene and EDTA were always included in the reaction mixtures, and no generation of TBARS was found in the H-LDL preparations (data not shown). The cells were then incubated with or without H-LDL, and the expression of different cytokine genes was analyzed using cDNA transcribed from isolated total RNA. Incubation of macrophages for 3 hours with H-LDL induced an upregulation of the mRNA expression of IL-8 in these cells ( Figure 1A and 1 B). After 24 hours, the expression of IL-8 mRNA returned back to basal levels ( Figure 1 C). H-LDL failed to induce upregulation of the tumor necrosis factor- (TNF- ), a pleiotropic proinflammatory cytokine that is upregulated by oxidized LDL. 12 The increased expression of IL-8 in H-LDL-treated macrophages was confirmed by RT-PCR, and was found to be time-dependent ( Figure 1 D).
Figure 1. Expression of human common cytokines in cultured vascular cells. Human monocyte-derived macrophages were incubated without (A) or with 50 µg/mL of H-LDL for 3 hours (B) or 24 hours (C). After incubations, total RNAs were extracted and expression levels of common human cytokines were analyzed with cDNA array. Upregulation of IL-8 gene by H-LDL was confirmed by RT-PCR (D). cDNAs were prepared, amplified by PCR, and the products were analyzed on 1.4% agarose gel and quantified. The bars represent means of 6 independent experiments ±SEM. * P <0.05, ** P <0.01.
Next, secretion of IL-8 from the H-LDL-stimulated cells into the culture medium was analyzed as a function of time. We found that H-LDL (50 µg cholesterol/mL) strongly induced (by 30-fold) the secretion of IL-8 from macrophages when compared with untreated cells ( Figure 2 A). Native (untreated) LDL (25 µg protein/mL that equals to 50 µg of total cholesterol/mL) was used as an additional control and did not induce secretion of IL-8 from macrophages during the 24-hour incubation time ( Figure 2 B). H-LDL was also compared with other inflammatory agents, such as LPS and IL-1ß, and was found to be as potent inducer of IL-8 secretion from macrophages as these traditional activators of cytokine secretion (data not shown). Surprisingly, although both the cDNA array and the RT-PCR showed upregulation of IL-1ß in the H-LDL-treated cells, secretion of IL-1ß protein was barely detectable in the cell culture media, suggesting that secretion of IL-1ß was abolished by posttranscriptional regulation (data not shown). 13
Figure 2. Secretion of IL-8 by hydrolase-modified LDL (H-LDL)-stimulated macrophages. A, Human monocyte-derived macrophages were incubated for the indicated times with 50 µg/mL of H-LDL. After incubation, the cell culture media were analyzed for IL-8 with commercial ELISA kit. B, As an additional control, macrophages were incubated without or with H-LDL (50 µg cholesterol/mL) and native LDL (25 µg protein/mL) for 6 hours, and secretion of IL-8 into cell culture media were analyzed by ELISA. Data shown are means of at least 3 independent experiments ±SEM.
To study the signaling pathways of H-LDL-induced IL-8 secretion in the cultured macrophages in more detail, specific inhibitors of the three main MAPKs, ie, p38, extracellular signal regulated kinase (ERK) 1/2, and JNK were included in the incubations, as described in the Methods. The inhibitor of p38 (SB203580 ) was found to strongly inhibit the release of IL-8, whereas the inhibitors of ERK 1/2 (PD 98059) and of JNK (JNKi II) did not have significant effects on the secretion of IL-8 from the H-LDL-treated macrophages ( Figure 3 A), suggesting that secretion of IL-8 involves activation of p38 MAPK. By Western blot analysis, we found that H-LDL rapidly induced phosphorylation of p38 MAPK in macrophages, ie, after only of 5 minutes of incubation ( Figure 3 B).
Figure 3. Participation of MAPKs in secretion of IL-8 from H-LDL-treated macrophages. A, Macrophages were preincubated for 30 minutes in the presence or absence of the indicated inhibitors: SB203580 (p38 MAPK, 10 µmol/L), PD98059 (ERK1/2 MAPKs, 30 µmol/L), and JNKi II (JNK MAPK, 5 µmol/L). Then, 50 µg/mL of H-LDL was added and incubation was continued for 6 hours. After the incubation, the media were analyzed for IL-8 by ELISA. Data shown are means of 5 independent experiments ±SEM. P <0.05. B, Macrophages were stimulated for indicated times with H-LDL, the cellular proteins (50 µg) were run on 15% SDS-PAGE, transferred into PVDF membranes, and detected with appropriate antibodies, and visualized with enhanced chemiluminescence kit.
Because the promoter area of IL-8 contains binding sites for NF- B and activator protein 1 (AP-1) transcription factors, 14 and nuclear translocation of NF- B and AP-1 is needed for IL-8 secretion by cultured human monocytes, 15 activation of NF- B and AP-1 was analyzed in H-LDL-treated macrophages. As shown in Figure 4, H-LDL did induce nuclear translocation of the transcription factors NF- B and AP-1, the former being translocated after 120 minutes and the latter after 60 minutes of incubation. In addition, a supershift assays revealed that the NF- B heterodimer contained p65 (Rel A) and p50, whereas AP-1 consisted of c-Jun, JunB, and JunD (data not shown). To further study the role of NF- B and AP-1 in the release of IL-8, macrophages were treated in the presence of inhibitors of these transcription factors. Caffeic acid phenylethyl ester (CAPE) and sulfasalazine, both capable of inhibiting NF- B, were found to almost totally block the secretion of IL-8, whereas curcumin, an inhibitor of JNK and AP-1, did not have any significant effect on the secretion of IL-8 ( Figure 5 ). The inability of JNKi II ( Figure 3 ) and curcumin to inhibit H-LDL-induced IL-8 release suggests that JNK and AP-1 do not play significant roles in the release of IL-8 from H-LDL-treated macrophages. As additional controls, macrophages were incubated for 6 hours with H-LDL in the presence of SB203580 (an inhibitor of p38) and CAPE (an inhibitor of NF- B), and uptake of H-LDL was analyzed by thin layer chromatography. We found that neither of these inhibitors decreased loading of macrophages with cholesteryl esters, suggesting that they inhibited secretion of IL-8 during the first 6 hours of incubation by inhibiting H-LDL-mediated signaling rather than uptake of H-LDL (data not shown). Taken together, although AP-1 may play a role in the regulation of IL-8 transcription in macrophages, NF- B appears to be more important in the expression and release of IL-8 from H-LDL-treated macrophages.
Figure 4. Nuclear translocation of NF- B and AP-1. Macrophages were incubated for the indicated times with 50 µg/mL of H-LDL and nuclear proteins were extracted. Nuclear extracts (4 to 10 µg) were analyzed by the electrophoretic mobility shift assay method, using 32 P-labeled oligonucleotides specific for NF- B (A) and AP-1 (B). Nuclear extracts from macrophages which were treated for 60 minutes with LPS (5 µg/mL) served as controls. Data shown are representative of 4 independent experiments.
Figure 5. The effect of inhibitors of NF- B and AP-1 on IL-8 secretion by H-LDL-stimulated macrophages. Macrophages were preincubated for 30 minutes in the presence or absence of the indicated inhibitors: curcumin (AP-1, 10 µmol/L), CAPE (NF- B, 15 µg/mL), sulfasalazin (NF- B, 2 mmol/L). Then, 50 µg/mL of H-LDL was added and incubation was continued for 6 hours. After incubation, IL-8 in the incubation media was quantified by ELISA. Data shown are means of 5 independent experiments ±SEM. * P <0.05.
Discussion
Our present results show that H-LDL, a modified LDL generated by hydrolases secreted by activated macrophages, 8 induces expression of proinflammatory cytokines, eg, IL-8, but not of anti-inflammatory cytokines, such as IL-10 and TGF-ß1. Interestingly, IL-8 expression and secretion appears to be one of the most prominent responses of macrophages, when activated by modified LDL(s). 2 IL-8 is a proatherosclerotic cytokine that has been shown to trigger firm adhesion of monocytes to the endothelial cells in vitro, 1 to modulate the extent of lesions in the arterial wall of genetically manipulated animals, 3 and to be expressed in the atherosclerotic lesions of human coronary arteries. 2
Comparison of the cellular effects of H-LDL with those of other LDL modifications shows both similarities and differences in the secretion pattern of cytokines and in the activation of signaling pathways. Oxidized LDL (oxLDL), the most studied modification of LDL, has been shown to induce secretion of not only IL-8, 2,5,16 but also IL-1ß, 17 and TNF- 18 from monocyte-macrophages. Interestingly, E-LDL, an LDL modification that resembles H-LDL, has been shown to induce the expression of IL-1ß mRNA without secretion of the mature protein in macrophages. 19 Similarly, H-LDL induced substantial expression of IL-1ß mRNA in macrophages, but the mature translational product was barely detectable in the cell culture media after 24 hours of incubation, suggesting that IL-1ß was subjected to post-transcriptional regulation. However, in sharp contrast to E-LDL that has been shown to induce secretion of IL-8 by endothelial cells, 7 but not by macrophages, 19 H-LDL induced substantial secretion of IL-8 by human monocyte-derived macrophages. Interestingly, even native LDL has been shown to induce secretion of IL-8 by smooth muscle cells. 20 However, we (this study) and others 2 have found that native LDL does not induce secretion of IL-8 by macrophages revealing that LDL needs to be modified to act as pathophysiological triggers of macrophage activation.
The mitogen-activated protein kinase (MAPK) p38 and the transcription factor NF- B play essential roles in the regulation of inflammatory reactions. They also have an important role in the control of IL-8 synthesis 21 and become activated by several external stimuli, such as cytokines and modified LDLs. OxLDL 22 and isoprostane, 23 a lipid peroxidation product present in oxLDL, both have been shown to activate both p38 and ERK1/2 MAPKs in macrophages. On activation by isoprostane, the expression of IL-8 in macrophages was induced by a mechanism that did not involve NF- B. 23 Rather, the secretion of IL-8 from H-LDL-stimulated macrophages depended on the activation of both NF- B and p38 MAPK, but not of AP-1 transcription factor or ERK and JNK MAPKs. Analysis of nuclear translocation of transcription factors by electrophoretic mobility shift assay revealed that in addition to NF- B, the amount of AP-1 also increased in the nucleus of H-LDL-treated macrophages. However, neither inhibition of JNK nor AP-1 did have an effect on IL-8 release, suggesting that although activated, JNK/AP-1 signaling pathway is not essential for expression of IL-8 in the H-LDL-treated macrophages. It has been suggested 100-fold) needs a coordinate action of at least 3 different signal transduction pathways which cooperate to induce mRNA synthesis and suppress mRNA degradation. 24 Accordingly, by activating 2 signaling pathways, ie, transcription of IL-8 through NF- B and stabilization of IL-8 mRNA by p38 MAPK, 21 H-LDL induced a less dramatic (30-fold, 100-fold) increase in the secretion of IL-8 in macrophages.
As described, oxLDL and H-LDL induce activation of different signaling pathways. In addition, H-LDL was generated in the presence of EDTA and butylated hydroxytoluene and was not significantly oxidized. These findings strongly argue against the possibility that the effects of H-LDL on macrophages were caused by oxidation of H-LDL lipids. In addition, the major phospholipids of LDL, ie, phosphatidylcholine and sphingomyelin, are not degraded in H-LDL, 8 suggesting that secretion of IL-8 by macrophages was not induced by lysophospholipids in H-LDL. Rather, the biologically active components that have been generated by lysosomal acid lipase, ie, unesterified cholesterol and free fatty acids are likely responsible for the inflammatory activity of H-LDL. We have previously found that hydrolysis of cholesterol esters by lysosomal acid lipase generates unesterified cholesterol in H-LDL, and that when added to cultured macrophages, H-LDL leads to an increased content of unesterified cholesterol in them. 8 In fact, intracellular content of unesterified cholesterol has been shown to control the secretion of IL-8 2 and to activate both p38 and NF- B in macrophages. 25 Moreover, cholesterol has been shown to be the main component of lipoproteins to activate p38 MAPK 26 and to induce secretion of IL-8. 27 H-LDL is also enriched with free fatty acids that have previously been shown to be critical in expression of IL-8 7,28 and activation of the p38 MAPK 29 and NF- B 28 in endothelial cells. The effects of free fatty acids are likely to be mediated by PPAR, which is abundantly expressed in endothelial cells and drives the expression of IL-8 in these cells. 30 However, in contrast to endothelial cells, macrophages strongly express PPAR rather than PPAR, 30,31 and the ligands of PPAR have been shown to induce expression of IL-8 in human macrophages. 32 Yet involvement of PPAR in the H-LDL-induced cytokine secretion appears unlikely, because H-LDL effectively activated both NF- B and AP-1 whereas PPAR downregulates NF- B and AP-1 activation and the transcription of proinflammatory cytokines. 33 Thus, the actual mechanism for activation of IL-8 release from H-LDL-treated macrophages remains to be elucidated.
The group of Bhakdi has in recent years mimicked arterial LDL modification by enzymatic modification of LDL in vitro by a protease (trypsin) and cholesterol esterase, and provided evidence for the presence of E-LDL in the arterial wall with the aid of monoclonal antibodies. 6,34 Inspired by their work, we have hypothesized that enzymatic modification of LDL could take place in atherosclerotic lesions by the action of lysosomal hydrolases secreted mainly by macrophages, and have found some immunohistochemical evidence that supports this hypothesis. 8 Here, we used a human cell culture model (immunologically stimulated human macrophages) in which the whole repertoire of lysosomal hydrolases secreted by the macrophages are able to modify LDL. Using this model, we have been able to generate an in vitro modified LDL that resembles arterial LDL in many respects, including morphology, 8 ability to generate foam cells from macrophages and smooth muscle cell, 8 and the ability to induce secretion of proinflammatory cytokines (this study). Further studies are required to reveal the actual role of the hydrolytic modifications of LDL in the process of atherosclerosis. However, our present findings suggest that in humans with established atherosclerosis, protection of macrophages from activation would be a plausible target of treatment to prevent the vicious circle consisting of: (1) local secretion of lysosomal hydrolases; (2) subsequent modification of LDL by the hydrolases; and (3) local autocrine-paracrine stimulation of inflammation mediated by the action of the hydrolase-modified LDL. 35
Acknowledgments
The Wihuri Research Institute is maintained by the Jenny and Antti Wihuri Foundation. The excellent technical assistance of Leena Saikko, Mari Jokinen, Päivi Ihamuotila, Elina Kaperi, and Anna Oksaharju is gratefully acknowledged.
Disclosures
None.
【参考文献】
Gerszten RE, Garcia-Zepeda EA, Lim YC, Yoshida M, Ding HA, Gimbrone MA Jr, Luster AD, Luscinskas FW, Rosenzweig A. MCP-1 and IL-8 trigger firm adhesion of monocytes to vascular endothelium under flow conditions. Nature. 1999; 398: 718-723.
Wang N, Tabas I, Winchester R, Ravalli S, Rabbani LE, Tall A. Interleukin 8 is induced by cholesterol loading of macrophages and expressed by macrophage foam cells in human atheroma. J Biol Chem. 1996; 271: 8837-8842.
Boisvert WA, Santiago R, Curtiss LK, Terkeltaub RA. A leukocyte homologue of the IL-8 receptor CXCR-2 mediates the accumulation of macrophages in atherosclerotic lesions of LDL receptor-deficient mice. J Clin Invest. 1998; 101: 353-363.
Ylä-Herttuala S, Palinski W, Rosenfeld ME, Parthasarathy S, Carew TE, Butler S, Witztum JL. Evidence for the presence of oxidatively modified low density lipoprotein in atherosclerotic lesions of rabbit and man. J Clin Invest. 1989; 84: 1086-1095.
Terkeltaub R, Banka CL, Solan J, Santoro D, Brand K, Curtiss LK. Oxidized LDL induces monocytic cell expression of interleukin-8, a chemokine with T-lymphocyte chemotactic activity. Arterioscler Thromb. 1994; 14: 47-53.
Torzewski M, Klouche M, Hock J, Messner M, Dorweiler B, Torzewski J, Gabbert HE, Bhakdi S. Immunohistochemical demonstration of enzymatically modified human LDL and its colocalization with the terminal complement complex in the early atherosclerotic lesion. Arterioscler Thromb Vasc Biol. 1998; 18: 369-378.
Suriyaphol P, Fenske D, Zahringer U, Han SR, Bhakdi S, Husmann M. Enzymatically modified nonoxidized low-density lipoprotein induces interleukin-8 in human endothelial cells: role of free fatty acids. Circulation. 2002; 106: 2581-2587.
Hakala JK, Oksjoki R, Laine P, Du H, Grabowski GA, Kovanen PT, Pentikäinen MO. Lysosomal enzymes are released from cultured human macrophages, hydrolyze LDL in vitro, and are present extracellularly in human atherosclerotic lesions. Arterioscler Thromb Vasc Biol. 2003; 23: 1430-1436.
Andrews NC, Faller DV. A rapid micropreparation technique for extraction of DNA-binding proteins from limiting numbers of mammalian cells. Nucleic Acids Res. 1991; 19: 2499.
Pentikäinen V, Suomalainen L, Erkkilä K, Martelin E, Parvinen M, Pentikäinen MO, Dunkel L. Nuclear factor-kappa B activation in human testicular apoptosis. Am J Pathol. 2002; 160: 205-218.
Hessler JR, Morel DW, Lewis LJ, Chisolm GM. Lipoprotein oxidation and lipoprotein-induced cytotoxicity. Arterioscler. 1983; 3: 215-222.
Miller YI, Viriyakosol S, Worrall DS, Boullier A, Butler S, Witztum JL. Toll-like receptor 4-dependent and -independent cytokine secretion induced by minimally oxidized low-density lipoprotein in macrophages. Arterioscler Thromb Vasc Biol. 2005; 25: 1213-1219.
Herzyk DJ, Allen JN, Marsh CB, Wewers MD. Macrophage and monocyte IL-1 beta regulation differs at multiple sites. Messenger RNA expression, translation, and post-translational processing. J Immunol. 1992; 149: 3052-3058.
Mukaida N, Okamoto S, Ishikawa Y, Matsushima K. Molecular mechanism of interleukin-8 gene expression. J Leukoc Biol. 1994; 56: 554-558.
Jefferson KK, Smith MF, Jr., Bobak DA. Roles of intracellular calcium and NF-kappa B in the Clostridium difficile toxin A-induced up-regulation and secretion of IL-8 from human monocytes. J Immunol. 1999; 163: 5183-5191.
Woenckhaus C, Kaufmann A, Bussfeld D, Gemsa D, Sprenger H, Grone HJ. Hypochlorite-modified LDL: chemotactic potential and chemokine induction in human monocytes. Clin Immunol Immunopathol. 1998; 86: 27-33.
Ku G, Thomas CE, Akeson AL, Jackson RL. Induction of interleukin 1 beta expression from human peripheral blood monocyte-derived macrophages by 9-hydroxyoctadecadienoic acid. J Biol Chem. 1992; 267: 14183-14188.
Janabi M, Yamashita S, Hirano K, Sakai N, Hiraoka H, Matsumoto K, Zhang Z, Nozaki S, Matsuzawa Y. Oxidized LDL-induced NF-kappa B activation and subsequent expression of proinflammatory genes are defective in monocyte-derived macrophages from CD36-deficient patients. Arterioscler Thromb Vasc Biol. 2000; 20: 1953-1960.
Klouche M, Gottschling S, Gerl V, Hell W, Husmann M, Dorweiler B, Messner M, Bhakdi S. Atherogenic properties of enzymatically degraded LDL: selective induction of MCP-1 and cytotoxic effects on human macrophages. Arterioscler Thromb Vasc Biol. 1998; 18: 1376-1385.
Ryoo SW, Kim DU, Won M, Chung KS, Jang YJ, Oh GT, Park SK, Maeng PJ, Yoo HS, Hoe KL. Native LDL induces interleukin-8 expression via H2O2, p38 Kinase, and activator protein-1 in human aortic smooth muscle cells. Cardiovasc Res. 2004; 62: 185-193.
Hoffmann E, Dittrich-Breiholz O, Holtmann H, Kracht M. Multiple control of interleukin-8 gene expression. J Leukoc Biol. 2002; 72: 847-855.
Senokuchi T, Matsumura T, Sakai M, Matsuo T, Yano M, Kiritoshi S, Sonoda K, Kukidome D, Nishikawa T, Araki E. Extracellular signal-regulated kinase and p38 mitogen-activated protein kinase mediate macrophage proliferation induced by oxidized low-density lipoprotein. Atherosclerosis. 2004; 176: 233-245.
Scholz H, Yndestad A, Damas JK, Waehre T, Tonstad S, Aukrust P, Halvorsen B. 8-isoprostane increases expression of interleukin-8 in human macrophages through activation of mitogen-activated protein kinases. Cardiovasc Res. 2003; 59: 945-954.
Holtmann H, Winzen R, Holland P, Eickemeier S, Hoffmann E, Wallach D, Malinin NL, Cooper JA, Resch K, Kracht M. Induction of interleukin-8 synthesis integrates effects on transcription and mRNA degradation from at least three different cytokine- or stress-activated signal transduction pathways. Mol Cell Biol. 1999; 19: 6742-6753.
Li Y, Schwabe RF, Devries-Seimon T, Yao PM, Gerbod-Giannone MC, Tall AR, Davis RJ, Flavell R, Brenner DA, Tabas I. Free cholesterol-loaded macrophages are an abundant source of tumor necrosis factor-{alpha} and interleukin-6: model of NF-{kappa}B- and MAP kinase-dependent inflammation in advanced atherosclerosis. J Biol Chem. 2005; 280: 21763-21772.
Dobreva I, Zschornig O, Waeber G, James RW, Widmann C. Cholesterol is the major component of native lipoproteins activating the p38 mitogen-activated protein kinases. Biol Chem. 2005; 386: 909-918.
Dobreva I, Waeber G, James RW, Widmann C. Interleukin-8 secretion by fibroblasts induced by low density lipoproteins is p38 MAPK-dependent and leads to cell spreading and wound closure. J Biol Chem. 2006; 281: 199-205.
Young VM, Toborek M, Yang F, McClain CJ, Hennig B. Effect of linoleic acid on endothelial cell inflammatory mediators. Metabolism. 1998; 47: 566-572.
Dersch K, Ichijo H, Bhakdi S, Husmann M. Fatty acids liberated from low-density lipoprotein trigger endothelial apoptosis via mitogen-activated protein kinases. Cell Death Differ. 2005; 12: 1107-1114.
Lee H, Shi W, Tontonoz P, Wang S, Subbanagounder G, Hedrick CC, Hama S, Borromeo C, Evans RM, Berliner JA, Nagy L. Role for peroxisome proliferator-activated receptor alpha in oxidized phospholipid-induced synthesis of monocyte chemotactic protein-1 and interleukin-8 by endothelial cells. Circ Res. 2000; 87: 516-521.
Nagy L, Tontonoz P, Alvarez JG, Chen H, Evans RM. Oxidized LDL regulates macrophage gene expression through ligand activation of PPARgamma. Cell. 1998; 93: 229-240.
Zhang X, Wang JM, Gong WH, Mukaida N, Young HA. Differential regulation of chemokine gene expression by 15-deoxy-delta 12,14 prostaglandin J2. J Immunol. 2001; 166: 7104-7111.
Ricote M, Li AC, Willson TM, Kelly CJ, Glass CK. The peroxisome proliferator-activated receptor-gamma is a negative regulator of macrophage activation. Nature. 1998; 391: 79-82.
Bhakdi S, Dorweiler B, Kirchmann R, Torzewski J, Weise E, Tranum J, Walev I, Wieland E. On the pathogenesis of atherosclerosis: enzymatic transformation of human low density lipoprotein to an atherogenic moiety. J Exp Med. 1995; 182: 1959-1971.
Pentikäinen MO, Öörni K, Ala-Korpela M, Kovanen PT. Modified LDL- trigger of atherosclerosis and inflammation in the arterial intima. J Intern Med. 2000; 247: 359-370.
作者单位：From Wihuri Research Institute, Helsinki, Finland.