Role of Leukotriene B4 Receptors in the Development of Atherosclerosis: Potential Mechanisms
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《动脉硬化血栓血管生物学》
From the James Graham Brown Cancer Center (K.S., V.R.J., B.H.) and the Departments of Microbiology and Immunology (S.M., J.S., B.H.), Medicine and Pharmacology & Toxicology (W.Z.), Anatomical Sciences & Neurobiology (M.T.T.), University of Louisville Health Sciences, Louisville, KY; and Department of Pathology (J.A., H.A.), University of Pennsylvania, School of Dental Medicine, Philadelphia, PA.
Correspondence to Dr Bodduluri Haribabu, Delia Baxter Bldg, University of Louisville, 580 South Preston Street, #119B, Louisville, KY 40202. E-mail H0bodd01@gwise.louisville.edu
Abstract
Objective— Leukotriene B4 (LTB4), a potent leukocyte chemoattractant, is known to promote several inflammatory diseases, including atherosclerosis. We sought to determine mechanisms through which LTB4 modulates atherosclerosis in cell lines expressing LTB4 receptors, BLT-1, and in mice deficient in BLT-1 as well as macrophage cell lines derived from BLT-1+/+ and BLT-1-/- mice.
Methods and Results— Analysis of global changes in gene expression induced by LTB4 in rat basophilic leukemia cells (RBL-2H3) expressing the human BLT-1 showed highest-fold increase in expression of fatty acid translocase/CD36 and the chemokine MCP1/JE/CCL2 , which are critical in atherogenesis. To determine the importance of BLT-1 in atherogenesis, we crossed BLT-1-null mice with apolipoprotein (apo)-E-deficient mice, which develop severe atherosclerosis. Deletion of BLT-1 significantly reduced the lesion formation in apo-E-/- mice only during initiating stages (4 and 8 weeks) but had no effect on the lesion size in mice fed atherogenic diet for 19 weeks. Macrophage cell lines from BLT-1-deficient mice expressed the low-affinity LTB4 receptor, BLT-2, and exhibited chemotaxis to LTB4.
Conclusions— The effects of LTB4 in atherosclerosis are likely mediated through the high-affinity BLT-1 and the low-affinity BLT-2 receptors. LTB4 promotes atherosclerosis by chemo-attracting monocytes, by providing an amplification loop of monocyte chemotaxis via CCL2 production, and by converting monocytes to foam cells by enhanced expression of CD36 and fatty acid accumulation.
Key Words: atherosclerosis ? monocyte/macrophages ? leukotriene B4 receptors
Introduction
Accumulation of monocytes in vascular subendothelial spaces and their conversion into lipid-laden "foam cells" is an early and important event in atherogenesis.1 The molecular mechanisms that regulate the recruitment of monocyte/macrophages to the vessel wall and the signaling pathways underlying their conversion to foam cells are poorly understood. Several recent studies suggest that chemokines CCL2/MCP1/JE, interleukin-8 (IL-8), and fractalkine and their receptors CCR2, CXCR2, and CX3CR1 are critical mediators of atherosclerosis.2–7 Mice lacking macrophage colony-stimulating factor have severely reduced atherosclerosis in experimental models.8 Thus, macrophages play a central role in the development of atherosclerotic vascular disease, which is now considered a chronic inflammatory disease.9
LTB4, a potent leukocyte chemoattractant, is known to promote a number of chronic inflammatory diseases.10 G-protein coupled receptors BLT-1 and BLT-2 and the peroxisome proliferator activator receptor (PPAR) are the currently known LTB4 receptors.11–13 While BLT-1 and BLT-2 likely mediate the proinflammatory responses of LTB4, PPAR, a transcription factor, might serve as a mediator of the anti-inflammatory effects of LTB4. Studies on mouse models and antagonists of LTB4 suggested a role for BLT-1 in rheumatoid arthritis, acute septic peritonitis, and atherosclerosis.14–16 To determine the role of BLT-1 in chronic inflammatory diseases, we analyzed LTB4-induced changes in global gene expression in cells expressing BLT-1 and atherosclerotic lesion development in BLT-1 and apolipoprotein-E (apo-E) double-deficient mice. We observed that several genes with known functions in the development of atherosclerosis are upregulated by LTB4, and deletion of BLT-1 gene offered early protection against development of atherosclerotic lesions in mice. Macrophage cell lines derived from BLT-1-deficient mice expressed a second LTB4 receptor, BLT-2. These cells also showed chemotaxis to higher LTB4 concentrations that are likely to occur in established atherosclerotic plaques. These results suggest that LTB4 is an important mediator and its receptors, BLT-1 and BLT-2, play critical and sequential roles during atherogenesis.
Methods
Analysis of LTB4-Induced Changes in Gene Expression
Global changes in LTB4-induced gene expression were determined in a rat basophilic leukemia cell line (RBL-2H3) expressing the human BLT-1 (hBLT-1) using the standard Affymetrix protocols and rat genome chip U34A (Affymetrix) containing probe sets for 8740 known rat genes. Changes in individual mRNAs were determined in real-time PCR experiments and individual proteins by flow cytometry or ELISA as detailed in supplemental data (see www.ahajournals.org).
Generation of BLT-1-/- and Apo-E-/- Mice
BLT-1-/- mice17,18 were backcrossed onto C57BL/6 background to the 8th generation. These mice were mated with apoE-/- mice also on the C57BL/6 background (Jackson Labs). The resulting offspring (BLT-1+/- and apoE+/-) were crossed with apo-E-/- mice and all the offspring were genotyped by standard PCR methods to select apo-E-/- and BLT-1+/- animals. These animals were setup as breeders and all 3 types of offspring, ie, BLT-1+/+, BLT-1+/-, and BLT-1-/- mice, in apo-E-/- background served as experimental animals. The double-knockout (apoE-/- BLT-1-/-) mice were born at the expected Mendelian ratios, developed normally, and were disease-free. Mice were weaned at 4 weeks, fed normal rodent chow (4.5% fat; Ralston Purina) for 3 more weeks, and switched to the western diet (21% fat, 0.15% cholesterol; Harlan Tekland no. 88137) at age 7 weeks.
Quantification of Atherosclerotic Lesions
Atherosclerotic lesions were analyzed using standard protocols with minor modifications.19,20 After collecting 0.5 mL whole blood, the anesthetized mice were transcardially perfused with saline followed by 4% buffered formalin. Aorta tree beginning at the aortic valve to the brachiocephalic artery, which is 2.5 mm, was removed and embedded in cryostat molds. Samples were snap-frozen by immersion in liquid nitrogen and stored at -20°C. The sectioning strategy was modified from that of Paigen et al20 as follows. The ascending segment from the appearance of aortic valve leaflets to the junction of the brachiocephalic artery, which is 1.5 mm, was sectioned. Then, 10-μm cryosections were serially made such that 2 contiguous sections were placed on 2 different slides. The next 5 sections were discarded. The cycle was repeated until the end of this segment. This method gave us at least 10 and up to 25 sections on each slide. One of the 2 slide sets was stained with Oil Red O stain as described.21 Images were examined in bright field using Nikon fluorescence microscope TE 300 and captured with digital color camera. Morphometric image analysis of the foamy–cell-laden atherosclerotic plaque was performed with Metamorph software version 5.0. For each animal, the average lesion area of 10 to 20 sections was determined and the data expressed as percent lesion area±SD. Representative sections from the other set were stained with MOMA-2 immunostain of the monocytes as described4, and the number of macrophages in lesions were counted (cells per high-power field). The remainder of the aorta was divided into the aortic arch, the thoracic aorta, and abdominal aorta for en face quantitative analysis of the atherosclerotic lesion by Sudan IV staining.16 The lesion area was expressed as percent lesion area of total area of pinned out arteries measured by digital morphometric analysis. Total plasma cholesterol and triglyceride levels were measured using commercially available kits (Sigma). Mice were fasted overnight before the collection of blood samples for lipid analysis. Statistical analyses of the lesion size data were performed using the nonparametric Mann-Whitney U test.
Generation and Analysis of Immortalized Macrophage Cell Lines
Immortalized wild-type and BLT-1-deficient murine macrophage cell lines were generated by J2 retroviral transformation as previously described.22 Details of phenotype, mRNA expression analysis, and methods for analysis of chemotaxis are provided in supplemental data (see www.ahajournals.org).
Results
LTB4-Induced Genes Involved in Atherosclerosis
Previous studies have shown that RBL cells expressing the hBLT-1 activate actin polymerization, pseudopod extension, chemotaxis, calcium mobilization, and degranulation in response to LTB4.23 To further assess the extent of cellular responses to LTB4, we analyzed changes in mRNA expression on Affymetrix rat U34A microarrays. Gene expression profiles in vehicle-treated hBLT-1-RBL cells or parental RBL cells treated with LTB4 were compared with LTB4-treated hBLT-1-RBL cells. The application of the double filter to data analysis resulted in a total of 17 upregulated genes, and the most striking observation from this analysis is that genes with well-established functions in the development of atherosclerosis showed highest-fold increases (Table 1). Fatty acid translocase/CD36 is a transmembrane protein and a specific receptor for oxidized LDL that transports LDL cholesterol into monocytes, thereby converting them to foam cells. Deletion of the CD36 gene in mice was protective against development of atherosclerotic lesions in otherwise susceptible mice.24 Rat JE (MCP-1/CCL2) is a ligand for the chemokine receptor CCR2 and a major regulator of monocyte/macrophage functions. CCL2, presumably produced at the site of vascular lesions, attracts monocytes to the area, and deletion of either CCL2 gene or its receptor CCR2 gene protected mice from developing atherosclerosis.2,4 Conversely, overexpression of CCL2 resulted in enhanced atherosclerosis in mice.25
Microarray Analysis of Changes in LTB4-induced Gene Expression
Among other genes specifically induced by LTB4 were urokinase plasminogen activator (uPA), colony-stimulating factor (CSF-1), and osteopontin. Although known to reduce vascular thrombosis, uPA was recently shown to significantly enhance experimental atherogenesis.26 CSF-1, the gene mutated in osteopetrotic mice, is essential for the development of atherosclerosis.27 A close examination of the microarray data also showed that osteopontin mRNA was upregulated 40-fold from parental RBL cells treated with LTB4 versus hBLT-1 cells treated with LTB4. Together, CSF-1 and osteopontin may be critical in promoting calcification and development of clinically significant aortic lesions.27,28 In addition, a number of other upregulated genes such as CD44, Src-like adapter protein (SLAP), and protein tyrosine phosphatase SHP-1, may have important functions in leukocyte recruitment and signal transduction. These results suggest that LTB4 is likely to have a major role in atherogenesis and provide targets for further analysis.
LTB4-induced upregulation of individual mRNAs and the corresponding proteins were tested in real-time PCR and protein expression experiments. The data (Figure 1) demonstrate that LTB4 enhanced the expression of mRNA and protein levels of CD36 (Figure 1A, 1C, and 1E) and CCL2 (Figure 1B, 1D,and 1F). Incubation of parental RBL cells with LTB4 did not result in any induction of either CD36 or CCL2. Moreover, the expression of both mRNAs and the corresponding proteins were dependent on Gi-protein signaling, as evidenced by near-complete inhibition of induction in cells treated with pertussis toxin. Pertussis toxin also inhibited the LTB4-induced upregulation of SLAP and uPA mRNAs (data not shown). A major difference between the expression of CD36 and CCL2 genes was that CCL2 induction appears to reach maximum levels both at lower doses of LTB4 and at earlier time points. This raises the possibility that some of the products of the genes induced early might promote upregulation of CD36. CSF-1 was one of the LTB4-induced genes (Table 1) and was known to induce the expression of CD36.29 However, addition of the medium from LTB4-treated hBLT1 cells (6 hours with 1 μmol/L) to RBL cells did not result in any detectable CD36 expression after an additional 18 hours, suggesting that LTB4-induced CD36 expression may be a direct effect of LTB4 on BLT-1 (data not shown). Therefore, LTB4 presumably activates distinct signaling pathways for the coordinated regulation of several genes involved in the development of atherosclerosis.
Figure 1. Analysis of LTB4-induced gene expression. Parental RBL cells or cells expressing human BLT-1 (hBLT-1) were treated as indicated and total RNA was isolated. Real-time PCR analysis was performed as described in Methods, and the data were expressed as fold induction over the levels in vehicle-treated versus LTB4-treated (1 μmol/L) cells for CD36 (A) and CCL2 (B) mRNAs. Cells were incubated with Pertussis toxin at 100 ng/mL overnight as indicated before the addition of LTB4. Time–course and dose–response of gene induction by LTB4 were determined in RBL or hBLT-1 cells. The indicated cell lines were treated with LTB4 (1.0 μmol/L) for different times (C and D) or different concentrations of LTB4 for 16 hours (E) or 6 hours (F), and the protein expression of CD36 (C and E) or CCL2 (D and F) were determined as described in Methods. The data in A–F are representative of at least 3 independent experiments, each with triplicate measurements.
BLT-1 Promotes Early Atherosclerotic Lesions in Apo-E-/--Deficient Mice
Given the dramatic induction of genes involved in atherogenesis by LTB4, we examined the involvement of BLT-1 in the development of atherosclerotic lesions in BLT-1-deficient mice. Deletion of BLT-1 is known to reduce LTB4-induced leukocyte adhesion under flow and inflammatory responses to thioglycolate or zymosan.17,18 Apo-E-/- mice spontaneously have lesions in the aortic valve and throughout arterial tree.4,30,31 Therefore, we bred BLT-1-/- mice with apo-E-/- mice and generated BLT-1 and apo-E double-deficient mice. Quantitative analysis of atherosclerotic lesion development in these mice fed a high fat western diet showed significantly reduced lesions in BLT-1-/- mice compared with BLT-1+/+ mice (Figures 2 and 3). First, we examined the lesion development along the aorta in whole-mount en face preparations stained with Sudan IV (Figure 1). In mice fed western diet for 4 weeks, the average lesions were 5.6±2.3% of the total area in BLT-1+/+ and 3.5±1.7% of the total area in BLT-1-/- mice (P=0.019, Mann-Whitney test). Although a 30% reduction in mean lesion area was also observed in BLT-1-/- mice at 8 weeks, the data are not statistically significant (data not shown). Aortic lesions were also measured by two other independent methods, a quantitative Oil Red O staining21 of the fatty lesions in aortic cross sections and a qualitative immunohistochemical staining of macrophages with MOMA-2 antibody in the same areas.4 Staining with Oil Red O confirmed the significant decrease in the total lesion area in BLT-1-/- mice after 4 weeks on the high-fat diet (Figure 3A–C). BLT-1+/- showed intermediate lesions both in en face and aortic cross-section analyses, suggesting a gene dosage effect. Typical MOMA-2 staining shown in Figure 3D and 3E indicates reduced monocyte infiltration in the lesions of the BLT-1-/- mice relative to BLT-1+/+ mice. However, when the mice were continued on the high-fat diet for 19 weeks, the lesion size was similar in BLT-1+/+ and BLT-1-/- mice as determined both in en face and aortic cross-section analyses (Figure I, available at http://atvb.ahajournals.org). Cholesterol and triglyceride levels in plasma samples of these mice were measured (Table I, available at http://atvb.ahajournals.org). While some variation was observed in these values, there is no clear pattern of changes in plasma lipid levels between BLT-1+/+ and BLT-1-/- animals.
Figure 2. Total lesion coverage in aortas of mice. A, The whole aorta divided into the aortic arch, the thoracic aorta, and abdominal aortas from mice fed high-fat diet for 4 or 8 weeks were mounted en face and stained for lipid with Sudan IV. Total lesion area in each of these segments was measured by digital morphometry. Each symbol represents a single animal and the bar represents means. The percentage of the lesion areas in BLT-1+/+ mice (n=8) at 4 weeks is significantly higher than in BLT-1-/- (n=8) mice (P=0.019, Mann–Whitney test; P=0.005, ANOVA). B, Representative sections of en face preparations stained with Sudan IV from BLT-1-/- and BLT-1+/+ mice.
Figure 3. Effect of BLT-1 deletion on lesions. A, Aortic cross sections (10 μm) were stained for lipid with Oil Red O and quantitated by digital morphometry. Each symbol represents an animal and the bars represent means for comparisons of BLT-1-/- and BLT-1+/+ mice at 4 weeks (P=0.036) and at 8 weeks (P=0.085, Mann–Whitney test). The difference in lesions between BLT-1+/+ and BLT-1+/- mice at 8 weeks is significant (P=0.041 Mann–Whitney test). Representative Oil Red O-stained sections of BLT-1+/- (B) and BLT-1+/+ (C), original magnification x200. Sections of aortic sinus stained for macrophages with MOMA-2 antibody (brown) and counter stained with hematoxylin from BLT-1-/- (D) and BLT-1+/+ (E) mice.
Macrophages From BLT-1-/- Mice Express a Functional BLT-2 Receptor
To analyze the expression of different leukotriene receptors and their signal transduction pathways in macrophages, the predominant leukocyte implicated in atherosclerosis, we generated immortalized macrophage cell lines from both BLT-1+/+ and BLT-1-/- cells.22 J2 retroviral transformation of murine bone marrow has been shown to result in the selective immortalization of cell lines that display the phenotypic and functional characteristics of macrophages, including LPS responsiveness and production of inflammatory cytokines, and nitric oxide production.32 Phenotypic analysis showed both of these cell lines expressed the typical macrophage marker CD11b and no T and B cell markers (Figure II, available at http://atvb.ahajournals.org). Real-time PCR analyses showed that both BLT-1 and BLT-2 are expressed in macrophages derived from BLT-1+/+ mice. More importantly, macrophages from BLT-1-/- mice showed the absence of BLT-1 transcripts but normal BLT-2 expression (Figure 4A). BLT-1+/+ macrophages showed robust chemotactic response with a maximum response at 1.0 nM LTB4. The BLT-1-/- cell line also showed chemotaxis but required a 100-fold higher LTB4 concentration for maximal response (Figure 4B and 4C). Both cell lines showed identical chemotactic responses to platelet activating factor (Figure III, available at http://atvb.ahajournals.org).
Figure 4. Expression and function of BLT-1 and BLT-2 in murine macrophages: A, Levels of BLT-1 and BLT-2 mRNAs in murine macrophage cell lines from BLT-1+/+ and BLT-1-/- mice were measured as described in Methods. The chemotaxis of BLT-1+/+ (B) and BLT-1-/- (C) macrophages to LTB4 is determined in transmembrane chemotaxis assays. The data shown are representative of three independent experiments.
Discussion
The results presented here point to a complex role for LTB4 and the two G-protein coupled receptors, BLT-1 and BLT-2, in atherosclerosis. Several recent reports support the view that LTB4 may play a critical role in atherosclerosis. High levels of all components involved in LTB4 biosynthesis, ie, 5-lipoxygenase, 5-lipoxygenase activating protein, and LTA4 hydrolase, were detected in human atherosclerotic lesions.33 Antagonists of LTB4 blocked the development of atherosclerosis in apo-E-deficient and LDLR-deficient mice,16 and mice deficient in 5-lipoxygenase showed greatly reduced lesions in LDLR-/- background, suggesting leukotrienes may play a dominant role in atherogenesis.34 However, the mechanisms through which LTB4 could bring about such dramatic differences are unclear. The data presented here suggest several mechanisms through which LTB4 might regulate atherogenesis (Figure IV, available at http://atvb.ahajournals.org). First, as a chemoattractant of monocytes, LTB4 could attract monocytes to the lipid-rich subendothelial spaces where it might be produced through the activation of 5-lipoxygenase. Alternatively, non-enzymatic lipid peroxidation produces isoleukotrienes that modulate biological processes through the activation of leukotriene receptors.35 Second, after the recruitment of monocytes to the developing atherosclerotic lesion, LTB4 can upregulate CCL2, CD36, uPA, CSF-1, and osteopontin, all of which have well-established roles at various stages during the development of atherosclerosis.2,4,24,26–28 In particular, the induction of CCL2 by LTB4 provides a positive feedback loop to recruit macrophages and further generation of LTB4 by CCL2-mediated activation of arachidonic acid metabolism.15 Likewise, the induction of CD36 by LTB4 provides yet another positive feedback loop via OxLDL uptake, conversion of macrophages to foam cells, and to the production of additional chemokines by OxLDL. Although multiple regulatory mechanisms for the expression of these individual genes are known, the coordinated regulation of all of these molecules through the activation of BLT-1 by LTB4 offers a clear initiating step in atherogenesis. We have not detected changes in CD36 and/or CCL2 mRNA levels in response to LTB4 in primary macrophages or macrophage lines. This could be because of the activation state and expression levels of BLT-1 in these cells. Resting macrophages express low levels of BLT-1, and activation was shown to increase BLT-1 mRNA levels.36 In addition, heterogeneous nature of monocyte/macrophages with subsets of cells expressing distinct markers was also reported.37 The macrophages in the atherosclerotic plaque are likely to be different from those in circulation or elicited by inflammatory mediators in peritoneal exudates.
The partial protection against lesion development in BLT-1-deficient mice at early times and no reduction in lesions in mice on prolonged high-fat diet is in contrast to the significant protection observed in mice treated with BLT-1 antagonist.16 This suggests that compensatory mechanisms for the loss of BLT-1 might have occurred in BLT-1-deficient mice. These may include upregulation of other chemokines, like MIP-2, KC, CX3CL1, and possibly CCL-2, acting through CXCR2, CX3CR1, and CCR2 receptors (all demonstrated to be involved in macrophage influx during atherogenesis).2–4 In addition, the nearly complete protection against atherogenesis offered by reduced 5-lipoxygenase activity34 suggests that other leukotrienes (LTC4 and LTD4) and leukotriene receptors (Cys LTs and BLT-2) are likely to be critical mediators of lesion development. Indeed, the results presented here showed that macrophages from BLT-1-deficient mice expressed the low-affinity LTB4 receptor BLT-2 and chemotactic response to LTB4. Cys LT and BLT-2 were also shown to be expressed on macrophages as well as in endothelial cells.38 The studies with LTB4 antagonists are also in agreement with a late role for BLT-2, because there was a greater protection at early times (< 70%) but reduced effectiveness at later times (>25%).16 Because established atherosclerotic plaques have very high levels of 5-lipoxygenase pathway components,33 it is likely that high local LTB4 concentrations that are inhibitory for chemotaxis through BLT-1 will be generated at these sites. Presence of a low-affinity BLT-2 on monocytes will allow these cells to migrate to the lesions at high LTB4 concentrations. Such tandem of high- and low-affinity chemoattractant receptors have been shown to mediate chemotaxis at different concentrations of the ligand.39 Demonstration of a more direct role for BLT-2 in atherosclerosis requires the development of BLT-2-specific antagonists and generation of BLT-2-/- mice.
The results presented herein suggest that LTB4 and its receptors BLT-1 and BLT-2 might play distinct roles in the initiation and progression of atherosclerotic disease. Selective interference with either LTB4 synthesis or function of BLT-1 and BLT-2 offers attractive targets for the development of pharmacological agents to block the progression of atherosclerotic vascular disease.
Acknowledgments
We thank J. Eaton for critical readings of the manuscript. Technical assistance was provided by Sabine J. Weigel in microarray analysis and Barbara Kalinowska in cryosectioning and histology. We also thank Bharati Matta for generating macrophage cell lines from mice. This research was supported by NIH grants AI-43184 and AI-52381 to B.H.
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Toda A, Yokomizo T, Masuda K, Nakao A, Izumi T, Shimizu T. Cloning and characterization of rat leukotriene B-4 receptor. Biochem Biophys Res Commun. 1999; 262: 806–812.
Geissmann F, Jung S, Littman DR. Blood monocytes consist of two principal subsets with distinct migratory properties. Immunity. 2003; 19: 71–82.
Lotzer K, Spanbroek R, Hildner M, Urbach A, Heller R, Bretschneider E, Galczenski H, Evans JF, Habenicht AJ. Differential leukotriene receptor expression and calcium responses in endothelial cells and macrophages indicate 5-lipoxygenase-dependent circuits of inflammation and atherogenesis. Arterioscler Thromb Vasc Biol. 2003; 23: E32–E36.
Hartt JK, Barish G, Murphy PM, Gao JL. N-formylpeptides induce two distinct concentration optima for mouse neutrophil chemotaxis by differential interaction with two N-formylpeptide receptor (FPR) subtypes. Molecular characterization of FPR2, a second mouse neutrophil FPR. J Exp Med. 1999; 190: 741–747.(Krishnaprasad Subbarao; V)
Correspondence to Dr Bodduluri Haribabu, Delia Baxter Bldg, University of Louisville, 580 South Preston Street, #119B, Louisville, KY 40202. E-mail H0bodd01@gwise.louisville.edu
Abstract
Objective— Leukotriene B4 (LTB4), a potent leukocyte chemoattractant, is known to promote several inflammatory diseases, including atherosclerosis. We sought to determine mechanisms through which LTB4 modulates atherosclerosis in cell lines expressing LTB4 receptors, BLT-1, and in mice deficient in BLT-1 as well as macrophage cell lines derived from BLT-1+/+ and BLT-1-/- mice.
Methods and Results— Analysis of global changes in gene expression induced by LTB4 in rat basophilic leukemia cells (RBL-2H3) expressing the human BLT-1 showed highest-fold increase in expression of fatty acid translocase/CD36 and the chemokine MCP1/JE/CCL2 , which are critical in atherogenesis. To determine the importance of BLT-1 in atherogenesis, we crossed BLT-1-null mice with apolipoprotein (apo)-E-deficient mice, which develop severe atherosclerosis. Deletion of BLT-1 significantly reduced the lesion formation in apo-E-/- mice only during initiating stages (4 and 8 weeks) but had no effect on the lesion size in mice fed atherogenic diet for 19 weeks. Macrophage cell lines from BLT-1-deficient mice expressed the low-affinity LTB4 receptor, BLT-2, and exhibited chemotaxis to LTB4.
Conclusions— The effects of LTB4 in atherosclerosis are likely mediated through the high-affinity BLT-1 and the low-affinity BLT-2 receptors. LTB4 promotes atherosclerosis by chemo-attracting monocytes, by providing an amplification loop of monocyte chemotaxis via CCL2 production, and by converting monocytes to foam cells by enhanced expression of CD36 and fatty acid accumulation.
Key Words: atherosclerosis ? monocyte/macrophages ? leukotriene B4 receptors
Introduction
Accumulation of monocytes in vascular subendothelial spaces and their conversion into lipid-laden "foam cells" is an early and important event in atherogenesis.1 The molecular mechanisms that regulate the recruitment of monocyte/macrophages to the vessel wall and the signaling pathways underlying their conversion to foam cells are poorly understood. Several recent studies suggest that chemokines CCL2/MCP1/JE, interleukin-8 (IL-8), and fractalkine and their receptors CCR2, CXCR2, and CX3CR1 are critical mediators of atherosclerosis.2–7 Mice lacking macrophage colony-stimulating factor have severely reduced atherosclerosis in experimental models.8 Thus, macrophages play a central role in the development of atherosclerotic vascular disease, which is now considered a chronic inflammatory disease.9
LTB4, a potent leukocyte chemoattractant, is known to promote a number of chronic inflammatory diseases.10 G-protein coupled receptors BLT-1 and BLT-2 and the peroxisome proliferator activator receptor (PPAR) are the currently known LTB4 receptors.11–13 While BLT-1 and BLT-2 likely mediate the proinflammatory responses of LTB4, PPAR, a transcription factor, might serve as a mediator of the anti-inflammatory effects of LTB4. Studies on mouse models and antagonists of LTB4 suggested a role for BLT-1 in rheumatoid arthritis, acute septic peritonitis, and atherosclerosis.14–16 To determine the role of BLT-1 in chronic inflammatory diseases, we analyzed LTB4-induced changes in global gene expression in cells expressing BLT-1 and atherosclerotic lesion development in BLT-1 and apolipoprotein-E (apo-E) double-deficient mice. We observed that several genes with known functions in the development of atherosclerosis are upregulated by LTB4, and deletion of BLT-1 gene offered early protection against development of atherosclerotic lesions in mice. Macrophage cell lines derived from BLT-1-deficient mice expressed a second LTB4 receptor, BLT-2. These cells also showed chemotaxis to higher LTB4 concentrations that are likely to occur in established atherosclerotic plaques. These results suggest that LTB4 is an important mediator and its receptors, BLT-1 and BLT-2, play critical and sequential roles during atherogenesis.
Methods
Analysis of LTB4-Induced Changes in Gene Expression
Global changes in LTB4-induced gene expression were determined in a rat basophilic leukemia cell line (RBL-2H3) expressing the human BLT-1 (hBLT-1) using the standard Affymetrix protocols and rat genome chip U34A (Affymetrix) containing probe sets for 8740 known rat genes. Changes in individual mRNAs were determined in real-time PCR experiments and individual proteins by flow cytometry or ELISA as detailed in supplemental data (see www.ahajournals.org).
Generation of BLT-1-/- and Apo-E-/- Mice
BLT-1-/- mice17,18 were backcrossed onto C57BL/6 background to the 8th generation. These mice were mated with apoE-/- mice also on the C57BL/6 background (Jackson Labs). The resulting offspring (BLT-1+/- and apoE+/-) were crossed with apo-E-/- mice and all the offspring were genotyped by standard PCR methods to select apo-E-/- and BLT-1+/- animals. These animals were setup as breeders and all 3 types of offspring, ie, BLT-1+/+, BLT-1+/-, and BLT-1-/- mice, in apo-E-/- background served as experimental animals. The double-knockout (apoE-/- BLT-1-/-) mice were born at the expected Mendelian ratios, developed normally, and were disease-free. Mice were weaned at 4 weeks, fed normal rodent chow (4.5% fat; Ralston Purina) for 3 more weeks, and switched to the western diet (21% fat, 0.15% cholesterol; Harlan Tekland no. 88137) at age 7 weeks.
Quantification of Atherosclerotic Lesions
Atherosclerotic lesions were analyzed using standard protocols with minor modifications.19,20 After collecting 0.5 mL whole blood, the anesthetized mice were transcardially perfused with saline followed by 4% buffered formalin. Aorta tree beginning at the aortic valve to the brachiocephalic artery, which is 2.5 mm, was removed and embedded in cryostat molds. Samples were snap-frozen by immersion in liquid nitrogen and stored at -20°C. The sectioning strategy was modified from that of Paigen et al20 as follows. The ascending segment from the appearance of aortic valve leaflets to the junction of the brachiocephalic artery, which is 1.5 mm, was sectioned. Then, 10-μm cryosections were serially made such that 2 contiguous sections were placed on 2 different slides. The next 5 sections were discarded. The cycle was repeated until the end of this segment. This method gave us at least 10 and up to 25 sections on each slide. One of the 2 slide sets was stained with Oil Red O stain as described.21 Images were examined in bright field using Nikon fluorescence microscope TE 300 and captured with digital color camera. Morphometric image analysis of the foamy–cell-laden atherosclerotic plaque was performed with Metamorph software version 5.0. For each animal, the average lesion area of 10 to 20 sections was determined and the data expressed as percent lesion area±SD. Representative sections from the other set were stained with MOMA-2 immunostain of the monocytes as described4, and the number of macrophages in lesions were counted (cells per high-power field). The remainder of the aorta was divided into the aortic arch, the thoracic aorta, and abdominal aorta for en face quantitative analysis of the atherosclerotic lesion by Sudan IV staining.16 The lesion area was expressed as percent lesion area of total area of pinned out arteries measured by digital morphometric analysis. Total plasma cholesterol and triglyceride levels were measured using commercially available kits (Sigma). Mice were fasted overnight before the collection of blood samples for lipid analysis. Statistical analyses of the lesion size data were performed using the nonparametric Mann-Whitney U test.
Generation and Analysis of Immortalized Macrophage Cell Lines
Immortalized wild-type and BLT-1-deficient murine macrophage cell lines were generated by J2 retroviral transformation as previously described.22 Details of phenotype, mRNA expression analysis, and methods for analysis of chemotaxis are provided in supplemental data (see www.ahajournals.org).
Results
LTB4-Induced Genes Involved in Atherosclerosis
Previous studies have shown that RBL cells expressing the hBLT-1 activate actin polymerization, pseudopod extension, chemotaxis, calcium mobilization, and degranulation in response to LTB4.23 To further assess the extent of cellular responses to LTB4, we analyzed changes in mRNA expression on Affymetrix rat U34A microarrays. Gene expression profiles in vehicle-treated hBLT-1-RBL cells or parental RBL cells treated with LTB4 were compared with LTB4-treated hBLT-1-RBL cells. The application of the double filter to data analysis resulted in a total of 17 upregulated genes, and the most striking observation from this analysis is that genes with well-established functions in the development of atherosclerosis showed highest-fold increases (Table 1). Fatty acid translocase/CD36 is a transmembrane protein and a specific receptor for oxidized LDL that transports LDL cholesterol into monocytes, thereby converting them to foam cells. Deletion of the CD36 gene in mice was protective against development of atherosclerotic lesions in otherwise susceptible mice.24 Rat JE (MCP-1/CCL2) is a ligand for the chemokine receptor CCR2 and a major regulator of monocyte/macrophage functions. CCL2, presumably produced at the site of vascular lesions, attracts monocytes to the area, and deletion of either CCL2 gene or its receptor CCR2 gene protected mice from developing atherosclerosis.2,4 Conversely, overexpression of CCL2 resulted in enhanced atherosclerosis in mice.25
Microarray Analysis of Changes in LTB4-induced Gene Expression
Among other genes specifically induced by LTB4 were urokinase plasminogen activator (uPA), colony-stimulating factor (CSF-1), and osteopontin. Although known to reduce vascular thrombosis, uPA was recently shown to significantly enhance experimental atherogenesis.26 CSF-1, the gene mutated in osteopetrotic mice, is essential for the development of atherosclerosis.27 A close examination of the microarray data also showed that osteopontin mRNA was upregulated 40-fold from parental RBL cells treated with LTB4 versus hBLT-1 cells treated with LTB4. Together, CSF-1 and osteopontin may be critical in promoting calcification and development of clinically significant aortic lesions.27,28 In addition, a number of other upregulated genes such as CD44, Src-like adapter protein (SLAP), and protein tyrosine phosphatase SHP-1, may have important functions in leukocyte recruitment and signal transduction. These results suggest that LTB4 is likely to have a major role in atherogenesis and provide targets for further analysis.
LTB4-induced upregulation of individual mRNAs and the corresponding proteins were tested in real-time PCR and protein expression experiments. The data (Figure 1) demonstrate that LTB4 enhanced the expression of mRNA and protein levels of CD36 (Figure 1A, 1C, and 1E) and CCL2 (Figure 1B, 1D,and 1F). Incubation of parental RBL cells with LTB4 did not result in any induction of either CD36 or CCL2. Moreover, the expression of both mRNAs and the corresponding proteins were dependent on Gi-protein signaling, as evidenced by near-complete inhibition of induction in cells treated with pertussis toxin. Pertussis toxin also inhibited the LTB4-induced upregulation of SLAP and uPA mRNAs (data not shown). A major difference between the expression of CD36 and CCL2 genes was that CCL2 induction appears to reach maximum levels both at lower doses of LTB4 and at earlier time points. This raises the possibility that some of the products of the genes induced early might promote upregulation of CD36. CSF-1 was one of the LTB4-induced genes (Table 1) and was known to induce the expression of CD36.29 However, addition of the medium from LTB4-treated hBLT1 cells (6 hours with 1 μmol/L) to RBL cells did not result in any detectable CD36 expression after an additional 18 hours, suggesting that LTB4-induced CD36 expression may be a direct effect of LTB4 on BLT-1 (data not shown). Therefore, LTB4 presumably activates distinct signaling pathways for the coordinated regulation of several genes involved in the development of atherosclerosis.
Figure 1. Analysis of LTB4-induced gene expression. Parental RBL cells or cells expressing human BLT-1 (hBLT-1) were treated as indicated and total RNA was isolated. Real-time PCR analysis was performed as described in Methods, and the data were expressed as fold induction over the levels in vehicle-treated versus LTB4-treated (1 μmol/L) cells for CD36 (A) and CCL2 (B) mRNAs. Cells were incubated with Pertussis toxin at 100 ng/mL overnight as indicated before the addition of LTB4. Time–course and dose–response of gene induction by LTB4 were determined in RBL or hBLT-1 cells. The indicated cell lines were treated with LTB4 (1.0 μmol/L) for different times (C and D) or different concentrations of LTB4 for 16 hours (E) or 6 hours (F), and the protein expression of CD36 (C and E) or CCL2 (D and F) were determined as described in Methods. The data in A–F are representative of at least 3 independent experiments, each with triplicate measurements.
BLT-1 Promotes Early Atherosclerotic Lesions in Apo-E-/--Deficient Mice
Given the dramatic induction of genes involved in atherogenesis by LTB4, we examined the involvement of BLT-1 in the development of atherosclerotic lesions in BLT-1-deficient mice. Deletion of BLT-1 is known to reduce LTB4-induced leukocyte adhesion under flow and inflammatory responses to thioglycolate or zymosan.17,18 Apo-E-/- mice spontaneously have lesions in the aortic valve and throughout arterial tree.4,30,31 Therefore, we bred BLT-1-/- mice with apo-E-/- mice and generated BLT-1 and apo-E double-deficient mice. Quantitative analysis of atherosclerotic lesion development in these mice fed a high fat western diet showed significantly reduced lesions in BLT-1-/- mice compared with BLT-1+/+ mice (Figures 2 and 3). First, we examined the lesion development along the aorta in whole-mount en face preparations stained with Sudan IV (Figure 1). In mice fed western diet for 4 weeks, the average lesions were 5.6±2.3% of the total area in BLT-1+/+ and 3.5±1.7% of the total area in BLT-1-/- mice (P=0.019, Mann-Whitney test). Although a 30% reduction in mean lesion area was also observed in BLT-1-/- mice at 8 weeks, the data are not statistically significant (data not shown). Aortic lesions were also measured by two other independent methods, a quantitative Oil Red O staining21 of the fatty lesions in aortic cross sections and a qualitative immunohistochemical staining of macrophages with MOMA-2 antibody in the same areas.4 Staining with Oil Red O confirmed the significant decrease in the total lesion area in BLT-1-/- mice after 4 weeks on the high-fat diet (Figure 3A–C). BLT-1+/- showed intermediate lesions both in en face and aortic cross-section analyses, suggesting a gene dosage effect. Typical MOMA-2 staining shown in Figure 3D and 3E indicates reduced monocyte infiltration in the lesions of the BLT-1-/- mice relative to BLT-1+/+ mice. However, when the mice were continued on the high-fat diet for 19 weeks, the lesion size was similar in BLT-1+/+ and BLT-1-/- mice as determined both in en face and aortic cross-section analyses (Figure I, available at http://atvb.ahajournals.org). Cholesterol and triglyceride levels in plasma samples of these mice were measured (Table I, available at http://atvb.ahajournals.org). While some variation was observed in these values, there is no clear pattern of changes in plasma lipid levels between BLT-1+/+ and BLT-1-/- animals.
Figure 2. Total lesion coverage in aortas of mice. A, The whole aorta divided into the aortic arch, the thoracic aorta, and abdominal aortas from mice fed high-fat diet for 4 or 8 weeks were mounted en face and stained for lipid with Sudan IV. Total lesion area in each of these segments was measured by digital morphometry. Each symbol represents a single animal and the bar represents means. The percentage of the lesion areas in BLT-1+/+ mice (n=8) at 4 weeks is significantly higher than in BLT-1-/- (n=8) mice (P=0.019, Mann–Whitney test; P=0.005, ANOVA). B, Representative sections of en face preparations stained with Sudan IV from BLT-1-/- and BLT-1+/+ mice.
Figure 3. Effect of BLT-1 deletion on lesions. A, Aortic cross sections (10 μm) were stained for lipid with Oil Red O and quantitated by digital morphometry. Each symbol represents an animal and the bars represent means for comparisons of BLT-1-/- and BLT-1+/+ mice at 4 weeks (P=0.036) and at 8 weeks (P=0.085, Mann–Whitney test). The difference in lesions between BLT-1+/+ and BLT-1+/- mice at 8 weeks is significant (P=0.041 Mann–Whitney test). Representative Oil Red O-stained sections of BLT-1+/- (B) and BLT-1+/+ (C), original magnification x200. Sections of aortic sinus stained for macrophages with MOMA-2 antibody (brown) and counter stained with hematoxylin from BLT-1-/- (D) and BLT-1+/+ (E) mice.
Macrophages From BLT-1-/- Mice Express a Functional BLT-2 Receptor
To analyze the expression of different leukotriene receptors and their signal transduction pathways in macrophages, the predominant leukocyte implicated in atherosclerosis, we generated immortalized macrophage cell lines from both BLT-1+/+ and BLT-1-/- cells.22 J2 retroviral transformation of murine bone marrow has been shown to result in the selective immortalization of cell lines that display the phenotypic and functional characteristics of macrophages, including LPS responsiveness and production of inflammatory cytokines, and nitric oxide production.32 Phenotypic analysis showed both of these cell lines expressed the typical macrophage marker CD11b and no T and B cell markers (Figure II, available at http://atvb.ahajournals.org). Real-time PCR analyses showed that both BLT-1 and BLT-2 are expressed in macrophages derived from BLT-1+/+ mice. More importantly, macrophages from BLT-1-/- mice showed the absence of BLT-1 transcripts but normal BLT-2 expression (Figure 4A). BLT-1+/+ macrophages showed robust chemotactic response with a maximum response at 1.0 nM LTB4. The BLT-1-/- cell line also showed chemotaxis but required a 100-fold higher LTB4 concentration for maximal response (Figure 4B and 4C). Both cell lines showed identical chemotactic responses to platelet activating factor (Figure III, available at http://atvb.ahajournals.org).
Figure 4. Expression and function of BLT-1 and BLT-2 in murine macrophages: A, Levels of BLT-1 and BLT-2 mRNAs in murine macrophage cell lines from BLT-1+/+ and BLT-1-/- mice were measured as described in Methods. The chemotaxis of BLT-1+/+ (B) and BLT-1-/- (C) macrophages to LTB4 is determined in transmembrane chemotaxis assays. The data shown are representative of three independent experiments.
Discussion
The results presented here point to a complex role for LTB4 and the two G-protein coupled receptors, BLT-1 and BLT-2, in atherosclerosis. Several recent reports support the view that LTB4 may play a critical role in atherosclerosis. High levels of all components involved in LTB4 biosynthesis, ie, 5-lipoxygenase, 5-lipoxygenase activating protein, and LTA4 hydrolase, were detected in human atherosclerotic lesions.33 Antagonists of LTB4 blocked the development of atherosclerosis in apo-E-deficient and LDLR-deficient mice,16 and mice deficient in 5-lipoxygenase showed greatly reduced lesions in LDLR-/- background, suggesting leukotrienes may play a dominant role in atherogenesis.34 However, the mechanisms through which LTB4 could bring about such dramatic differences are unclear. The data presented here suggest several mechanisms through which LTB4 might regulate atherogenesis (Figure IV, available at http://atvb.ahajournals.org). First, as a chemoattractant of monocytes, LTB4 could attract monocytes to the lipid-rich subendothelial spaces where it might be produced through the activation of 5-lipoxygenase. Alternatively, non-enzymatic lipid peroxidation produces isoleukotrienes that modulate biological processes through the activation of leukotriene receptors.35 Second, after the recruitment of monocytes to the developing atherosclerotic lesion, LTB4 can upregulate CCL2, CD36, uPA, CSF-1, and osteopontin, all of which have well-established roles at various stages during the development of atherosclerosis.2,4,24,26–28 In particular, the induction of CCL2 by LTB4 provides a positive feedback loop to recruit macrophages and further generation of LTB4 by CCL2-mediated activation of arachidonic acid metabolism.15 Likewise, the induction of CD36 by LTB4 provides yet another positive feedback loop via OxLDL uptake, conversion of macrophages to foam cells, and to the production of additional chemokines by OxLDL. Although multiple regulatory mechanisms for the expression of these individual genes are known, the coordinated regulation of all of these molecules through the activation of BLT-1 by LTB4 offers a clear initiating step in atherogenesis. We have not detected changes in CD36 and/or CCL2 mRNA levels in response to LTB4 in primary macrophages or macrophage lines. This could be because of the activation state and expression levels of BLT-1 in these cells. Resting macrophages express low levels of BLT-1, and activation was shown to increase BLT-1 mRNA levels.36 In addition, heterogeneous nature of monocyte/macrophages with subsets of cells expressing distinct markers was also reported.37 The macrophages in the atherosclerotic plaque are likely to be different from those in circulation or elicited by inflammatory mediators in peritoneal exudates.
The partial protection against lesion development in BLT-1-deficient mice at early times and no reduction in lesions in mice on prolonged high-fat diet is in contrast to the significant protection observed in mice treated with BLT-1 antagonist.16 This suggests that compensatory mechanisms for the loss of BLT-1 might have occurred in BLT-1-deficient mice. These may include upregulation of other chemokines, like MIP-2, KC, CX3CL1, and possibly CCL-2, acting through CXCR2, CX3CR1, and CCR2 receptors (all demonstrated to be involved in macrophage influx during atherogenesis).2–4 In addition, the nearly complete protection against atherogenesis offered by reduced 5-lipoxygenase activity34 suggests that other leukotrienes (LTC4 and LTD4) and leukotriene receptors (Cys LTs and BLT-2) are likely to be critical mediators of lesion development. Indeed, the results presented here showed that macrophages from BLT-1-deficient mice expressed the low-affinity LTB4 receptor BLT-2 and chemotactic response to LTB4. Cys LT and BLT-2 were also shown to be expressed on macrophages as well as in endothelial cells.38 The studies with LTB4 antagonists are also in agreement with a late role for BLT-2, because there was a greater protection at early times (< 70%) but reduced effectiveness at later times (>25%).16 Because established atherosclerotic plaques have very high levels of 5-lipoxygenase pathway components,33 it is likely that high local LTB4 concentrations that are inhibitory for chemotaxis through BLT-1 will be generated at these sites. Presence of a low-affinity BLT-2 on monocytes will allow these cells to migrate to the lesions at high LTB4 concentrations. Such tandem of high- and low-affinity chemoattractant receptors have been shown to mediate chemotaxis at different concentrations of the ligand.39 Demonstration of a more direct role for BLT-2 in atherosclerosis requires the development of BLT-2-specific antagonists and generation of BLT-2-/- mice.
The results presented herein suggest that LTB4 and its receptors BLT-1 and BLT-2 might play distinct roles in the initiation and progression of atherosclerotic disease. Selective interference with either LTB4 synthesis or function of BLT-1 and BLT-2 offers attractive targets for the development of pharmacological agents to block the progression of atherosclerotic vascular disease.
Acknowledgments
We thank J. Eaton for critical readings of the manuscript. Technical assistance was provided by Sabine J. Weigel in microarray analysis and Barbara Kalinowska in cryosectioning and histology. We also thank Bharati Matta for generating macrophage cell lines from mice. This research was supported by NIH grants AI-43184 and AI-52381 to B.H.
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