Interleukin-18 Enhances Atherosclerosis in Apolipoprotein E−/− Mice Through Release of Interferon-γ
We have previously shown that interferon-γ (IFN-γ) is a potent enhancer of atherogenesis. Interleukin-18 (IL-18) promotes inflammatory responses through release of IFN-γ, although it can also exert direct actions on other inflammatory mediators. In this present study, we determined the effects of IL-18 on atherogenesis and the role of IFN-γ in this response. Male apolipoprotein E−/− mice (apoe−/−; aged 16 weeks, n=10/group) were fed a normal diet and injected intraperitoneally for 30 days with either recombinant IL-18 (30 ng/g/day) or saline. Atherosclerotic lesion size was quantified in 2 vascular beds: the ascending aorta and the aortic arch. IL-18 administration did not affect serum cholesterol concentrations or lipoprotein-cholesterol distribution; however, exogenous IL-18 administration increased lesion size 2-fold in both the ascending aorta (50 642±12 515 versus 112 399±13 227 μm2, P=0.004; saline versus IL-18 groups, respectively) and the aortic arch (3.1±0.3% versus 6.2±0.9% area, P=0.006). Exogenous IL-18 promoted a 4-fold increase in the number of lesion-associated T lymphocytes (11±3 versus 50±5 cells; P<0.0001) and cells expressing major histocompatability complex class II (9±3 versus 40±6 cells; P=0.0002). To determine the role of IFN-γ production in this response, exogenous IL-18 was administered to apoe−/− mice that were IFN-γ deficient. These studies demonstrated that lack of endogenous IFN-γ ablated the effects of IL-18 on atherosclerosis. Therefore, these data strongly implicates IL-18 in the atherogenic process and suggests that IL-18 increases lesion development through enhancement of an inflammatory response involving an IFN-γ–dependent mechanism. The full text of this article is available at http://www.circresaha.org.
Atherosclerosis is an inflammatory process involving the recruitment of specific leukocytes, especially macrophages and T lymphocytes.1–3⇓⇓ A wide array of soluble mediators has been proposed to regulate the inflammatory process.1,2⇓ Interferon-γ (IFN-γ) is emerging as a prominent proinflammatory mediator of atherosclerosis. Evidence for its role in the disease process includes the presence of IFN-γ mRNA and protein in atherosclerotic lesions of both humans4 and mice.5–7⇓⇓ Deficiency of either the IFN-γ receptor8 or the cytokine (S.C. Whitman, P. Ravisankar, P. Veeraraghavan, A. Daugherty, unpublished data, 2002) leads to a profound decrease in the severity of atherosclerosis in apolipoprotein E−/− (apoe−/−) mice. Conversely, the exogenous administration of recombinant IFN-γ promotes the atherogenic process.10 Therefore, the role of IFN-γ and its regulator have important consequences for atherogenesis.
One of the primary factors regulating IFN-γ secretion is interleukin-18 (IL-18). This cytokine is a 24-kDa, non-glycosylated polypeptide that lacks a classical signal sequence11–14⇓⇓⇓ and possesses a structure similar to IL-1β.12,15⇓ IL-18 is synthesized as an inactive propeptide that undergoes proteolytic cleavage by the enzyme caspase-1 to generate a mature, bioactive, 18-kDa molecule.12,14,16⇓⇓ In both the mature and propeptide forms, IL-18 shows 64% sequence identity from mouse to human.12 Cells known to express IL-18 include macrophages/Kupffer cells,11,17⇓ keratinocytes,18 glucocorticoid-secreting adrenal cortex cells,19 and osteoblasts.20 Because of its ability to stimulate IFN-γ release by both NK cells and T lymphocytes, IL-18 is considered to be a key cytokine in the processes of innate and acquired immunity. IL-18 stimulates IFN-γ production by initiating IFN-γ gene transcription via activation of the IFN-γ promoter in a process that requires c-jun binding to the AP-1 site.21
A role of IL-18 in atherosclerosis has been inferred following its identification in human atherosclerotic lesions.22 Furthermore, overexpression of the murine IL-18 binding protein, the endogenous inhibitor of IL-18, has been shown to reduce atherosclerotic lesion development in apoe−/− mice.23 Therefore, there is associative evidence to infer a role of IL-18 in the development of atherosclerosis. The present study presents the first direct evidence indicating that IL-18 is proatherogenic, by showing that administration of exogenous IL-18 will enhance the development of atherosclerosis in apoe−/− mice and that this cytokine increases lesion development through enhancement of an inflammatory response via an IFN-γ–dependent mechanism.
Materials and Methods
Lyophilized recombinant mouse IL-18 (Cat. No. B002-5, R&D Systems, Inc, Minneapolis, Minn) was reconstituted in solution A (sterile phosphate buffered saline [PBS] containing 0.1% bovine serum albumin), to prepare a stock concentration of 50 μg/mL, as recommended by the manufacturer.
Mice and Cytokine Injections
apoe−/− and ifn-γ−/− mice, backcrossed 10 times to the C57BL/6 strain, were obtained from the Jackson Laboratory (Bar Harbor, Maine). The ifn-γ−/− mice were bred to apoe−/− mice to generate compound-deficient mice. Polymerase chain reaction (PCR) was used to identify mice homozygous for both apoE and IFN-γ deficiency, as described previously.24,25⇓ Breeding pairs of apoe−/− and apoe−/−×ifn-γ−/− mice were established and equal numbers of siblings from the resulting litters were divided into 2 groups of 10 male apoe−/− mice and 2 groups of 10 male apoe−/−×ifn-γ−/− mice. At 16 weeks old, one group from each strain received daily peritoneal injections of IL-18 (30 ng/g body weight) diluted in PBS, while the second group received daily injections of solution A (to control for the presence of bovine serum albumin), diluted also in PBS. The dose of recombinant cytokine was based on previous studies in which prolonged IL-18 administration via the intraperitoneal route exerted biological affects. Doses of administered recombinant IL-18 have ranged from ≈3026 to ≈40027 ng/g body wt/day. Therefore, based on these published findings, we decided to use a dose that was at the lowest value cited in the most current literature. Daily injections were carried out for 30 days, during which time the mice were kept on a normal laboratory diet. Mice were housed in a pathogen-free facility, with all of the animal procedures having the full approval of the University of Kentucky Institutional Animal Care and Use Committee.
Lipid and Lipoproteins
At the end of the study, serum total cholesterol and triglyceride concentrations were determined with enzymatic assay kits (Wako Chemical Co). Lipoprotein cholesterol distributions were evaluated in individual serum samples (50 μL) from 5 mice in each group that were each resolved by size exclusion chromatography on a Superose 6 column as described previously.10
Acquisition of Tissues
At the end of the injection period, mice were anesthetized by intraperitoneal injection of ketamine plus xylazine (90+10 mg/kg body weight). Terminal blood samples were collected by puncture of the right ventricle, and mice were perfused with PBS. Hearts were separated from the aorta at the base, embedded in OCT, and frozen at −20°C. Aortic tissue was removed from the ascending aorta to the level of the diaphragm and fixed overnight at room temperature in freshly prepared 4% paraformaldehyde. The livers and spleens of each mouse were removed, weighed, and segments of the spleens were frozen in OCT and stored at −20°C.
Quantification of Atherosclerotic Lesions
Atherosclerotic lesion size was determined in 2 vascular beds, the ascending aorta and the aortic arch, using 2 well-validated techniques, as described previously.10,28⇓ Lesion size in the ascending aorta was determined from 4 Oil Red O–stained serial sections, cut 10-μm thick, and collected at 100-μm intervals, starting at the region where the aortic sinus becomes the ascending aorta, as described previously.10 Lesion area, defined as intimal tissue within the internal elastic lamina, was determined using Image-Pro software (Media Cybernetics) on images that were created using a Spot camera (Diagnostic Instruments). The mean lesion area derived from the 4 serial sections was taken as the mean lesion size for each animal. Lesion size in the aortic arch was measured from en face preparations of this region, and the percentage of intimal surface area covered by atherosclerotic lesions was determined for the aortic arch, as described previously.28
Immunocytochemistry was performed, as described previously,10 on serial sections of the ascending aorta adjacent to those stained with Oil Red O. The following reagents were used for immunostaining: an anti-mouse Thy1.2 monoclonal antibody (01011D, 7μg/mL; PharMingen) and an anti-mouse major histocompatability complex class II (MHC II) monoclonal antibody (LS-004-SN, 1:5 dilution; Biosource International). Species-specific biotinylated secondary antibodies and a biotin-avidin-peroxidase complex were subsequently incubated with tissues (Vectastain Elite ABC kit, Vector Laboratories). Immunoreactivity was visualized using the red chromogen, 3-amino-9-ethyl carbazole (Biomeda Corp).
Data analyses were performed using SigmaStat 2.03 software (SPSS Inc). Statistical analysis between groups was by Student’s t test after testing that the data complied with the constraints of parametric analysis. In instances where parametric analysis was not permissible, analysis between groups was conducted using the Mann-Whitney rank sum test. Values with P≤0.05 were considered statistically significant.
When compared with mice injected with saline, injection of exogenous IL-18 did not affect serum cholesterol concentrations (272±15 versus 253±17 mg/dL, respectively) or lipoprotein-cholesterol distributions (Figure 1). No changes in serum triglyceride concentrations were found between groups (data not shown).
Although serum lipid concentrations did not differ between the 2 groups, injection of exogenous IL-18 caused a 2.2-fold increase in atherosclerotic lesion size in the ascending aorta (50 642±12 515 versus 112 399±13 227 μm2, control and IL-18–injected, respectively, P=0.004; Figure 2A). Further analysis also revealed that injection of IL-18 caused a significant increase throughout the ascending aorta (P≤0.01; Figure 2B). To complement lesion analysis in the ascending aorta, the extent of atherosclerosis between the 2 groups was also analyzed in the aortic arch, where we found that compared with saline-injected mice, exogenous IL-18 caused a 2-fold increase in atherosclerosis (3.1±0.3 versus 6.2±0.9% area, P=0.006; Figure 2C).
In addition to measuring for differences in lesion size between the 2 groups, immunocytochemical analyses of these same lesions were conducted to examine for any potential changes in the inflammatory status of these lesions. From these analyses, we found that compared with mice injected with saline, IL-18 injections increased the mean number of lesion-associated T lymphocytes by 4.5-fold (11±3 versus 50±5 mean number of cells found in the lesions of the ascending aorta, P<0.0001; Figure 3A) and the mean number of cells expressing MHC II by 4.4-fold (9±3 versus 40±6 mean number of cells found in the lesions of the ascending aorta, P=0.0002; Figure 3B).
IL-18 is known to stimulate the production of IFN-γ from NK cells, T lymphocytes, and macrophages. Furthermore, we have previously shown that IFN- γ promotes atherosclerosis in apoe−/− mice.10 Therefore, we examined whether administration of exogenous IL-18 to apoe−/− mice promoted atherosclerosis via an IFN-γ–mediated process. To accomplish this, we repeated our IL-18 injection studies using apoe−/− mice that were deficient in IFN-γ. In this group of mice, serum total cholesterol and triglyceride concentrations or lipoprotein-cholesterol distribution did not differ between groups (data not shown). Furthermore, IL-18 injections did not significantly increase lesion size measured in the ascending aorta (23 558±11 133 versus 11 657±3860 μm2, P=0.306, controls versus IL-18–injected mice, respectively; Figure 4A) or the aortic arch (1.5±0.4 versus 0.8±0.3% area of intimal surface, P=0.150; Figure 4B). As described previously (Whitman et al, unpublished data), lesion development in saline-injected apoe−/−×ifn-γ−/−–null mice was reduced by approximately 50% compared with saline-injected, apoe−/−×ifn-γ+/+ mice (Figure 4 versus Figure 2).
In addition, when compared with mice injected with saline, the injection of IL-18 into IFN-γ–deficient mice did not affect the mean number of lesion-associated T lymphocytes (2±1 versus 2±1 mean number of cells found in the lesions of the ascending aorta; P=0.784) or the mean number of cells immunostaining positive for MHC II (5±3 versus 3±1 mean number of cells found in the lesions of the ascending aorta; P=0.381).
Recently, Mallat et al22 demonstrated that IL-18 (mRNA and protein) is expressed in human carotid atherosclerotic plaques retrieved by endarterectomy. Furthermore, this group defined that exogenous expression of the murine IL-18 binding protein (the endogenous inhibitor of IL-18) reduced atherosclerotic lesion development in apoe−/− mice.23 Both of these studies associatively imply that IL-18 is a proatherogenic cytokine. However, as important as these 2 studies are at bringing awareness of the potential contribution of IL-18 to the disease process, they fail to provide direct evidence that IL-18 is in fact enhancing lesion progression. The present study provides this needed proof that IL-18 will promote lesion development and goes on to show that IL-18 enhances atherosclerosis via the promotion of an inflammatory process at the site of lesion development and that this proinflammatory response requires the production of IFN-γ.
IL-18 by itself is generally a weak stimulator of IFN-γ release. However, it functions synergistically with other co-stimulators, particularly IL-12, to induce pronounced secretion of IFN-γ production by T cells,29,30⇓ NK cells,31 and macrophages.32 Therefore, as it relates to this study, in order for exogenous IL-18 to have promoted atherosclerosis via an IFN-γ–mediated mechanism, it would require that at least one co-stimulus to have been present within the early developing lesions. As it turns out, one such co-stimulus has been identified by at least 2 laboratories. Recently, work by Uyemura et al33 and Lee et al,6 have identified IL-12 in atherosclerotic lesions from both humans and mice, respectively. Furthermore, Lee et al6 have shown that administration of exogenous IL-12 to apoe−/− mice will promote lesion development.
Direct evidence exists for the secretion of IFN-γ within atherosclerotic lesions of humans33–36⇓⇓⇓ and apoe−/− mice.5,6⇓ This cytokine can influence the development of atherosclerosis through a number of mechanisms that have been demonstrated in cultured cells. These include effects on 15-lipoxygenase,37 lipoprotein-metabolism,38 lipoprotein-modification,39 lipoprotein-cell recognition,36,40,41⇓⇓ apoptosis,42 extracellular matrix synthesis,43 and macrophage ABCA1 expression.44 However, these diverse effects of IFN-γ on cultured cells render it difficult to predict whether IFN-γ would promote or retard atherogenesis. Recently we have demonstrated that IFN-γ promotes atherosclerosis in apoe−/− mice. We have shown that intraperitoneal administration of exogenous IFN-γ into apoe−/− mice promoted atherosclerosis.10 Conversely, deficiency of endogenous IFN-γ production in apoe−/− mice, via targeted deletion of the ifn-γ gene, decreased atherosclerosis (Whitman et al, unpublished data). In this latter study, compound-deficient mice were generated by crossing strain-matched ifn-γ−/− and apoe−/− mice and comparing them to apoe−/−×ifn-γ+/+ mice. IFN-γ deficiency did not affect serum cholesterol concentrations or lipoprotein-cholesterol distributions in any groups. Furthermore, IFN-γ deficiency had no effect on the extent of atherosclerosis in female mice in either the aortic root or aortic arch. In contrast, IFN-γ deficiency markedly decreased lesions size in male apoe−/− mice, highlighting a pronounced gender-specific effect with regard to this proinflammatory cytokine. In the present study, only male apoe−/− mice were used and so the effect of IL-18 on lesion development in female apoe−/− mice is not known. This gender-specific effect of IFN-γ on apoe−/− mice warrants future studies to determine whether endogenous and exogenous IL-18 exerts effects in female mice.
Thus, IFN-γ is a potent proatherogenic cytokine, and having shown its role in promoting lesion development, we have now started to turn our attention toward defining the cellular sources of IFN-γ and the signaling responsible for its release. In summary, the present study provides strong evidence that IL-18 is a proatherogenic cytokine. Furthermore, its potent proinflammatory effect during atherosclerotic lesions formation is dependent on the presence of IFN-γ.
We would like to acknowledge support from a Heart and Stroke Foundation of Canada Fellowship (S.C.W.), an American Heart Association (Ohio Valley Affiliate) Fellowship (S.C.W), and an American Heart Association National Center Grant-in-Aid (A.D). The authors would like to thank Debra Rateri for technical assistance.
Original received November 30, 2001; revision received January 2, 2002; accepted January 8, 2002.
- ↵Daugherty A, Hansson GK. Lymphocytes in atherogenesis.In: Dean RT, Kelly D, eds. Atherosclerosis. New York, NY: Oxford Press; 2000: 230–249.
- ↵Lee TS, Yen HC, Pan CC, Chau LY. The role of interleukin 12 in the development of atherosclerosis in ApoE-deficient mice. Arterioscler Thromb Vasc Biol. 1999; 19: 734–742.
- ↵Huber SA, Sakkinen P, David C, Newell MK, Tracy RP. T helper–cell phenotype regulates atherosclerosis in mice under conditions of mild hypercholesterolemia. Circulation. 2001; 103: 2610–2616.
- Deleted in proof.
- ↵Ushio S, Namba M, Okura T, Hattori K, Nukada Y, Akita K, Tanabe F, Konishi K, Micallef M, Fujii M, Torigoe K, Tanimoto T, Fukuda S, Ikeda M, Okamura H, Kurimoto M. Cloning of the cDNA for human IFN-γ–inducing factor, expression in Escherichia coli, and studies on the biologic activities of the protein. J Immunol. 1996; 156: 4274–4279.
- ↵Ho AS, Liu Y, Khan TA, Hsu DH, Bazan JF, Moore KW. A receptor for interleukin 10 is related to interferon receptors. Proc Natl Acad Sci U S A. 1993; 90: 11267–11271.
- ↵Gu Y, Kuida K, Tsutsui H, Ku G, Hsiao K, Fleming MA, Hayashi N, Higashino K, Okamura H, Nakanishi K, Kurimoto M, Tanimoto T, Flavell RA, Sato V, Harding MW, Livingston DJ, Su MS. Activation of interferon-γ inducing factor mediated by interleukin-1β converting enzyme. Science. 1997; 275: 206–209.
- ↵Matsui K, Yoshimoto T, Tsutsui H, Hyodo Y, Hayashi N, Hiroishi K, Kawada N, Okamura H, Nakanishi K, Higashino K. Propionibacterium acnes treatment diminishes CD4+NK1.1+T cells but induces type I T cells in the liver by induction of IL-12 and IL-18 production from Kupffer cells. J Immunol. 1997; 159: 97–106.
- ↵Stoll S, Muller G, Kurimoto M, Saloga J, Tanimoto T, Yamauchi H, Okamura H, Knop J, Enk AH. Production of IL-18 (IFN-γ–inducing factor) messenger RNA and functional protein by murine keratinocytes. J Immunol. 1997; 159: 298–302.
- ↵Torigoe K, Ushio S, Okura T, Kobayashi S, Taniai M, Kunikata T, Murakami T, Sanou O, Kojima H, Fujii M, Ohta T, Ikeda M, Ikegami H, Kurimoto M. Purification and characterization of the human interleukin-18 receptor. J Biol Chem. 1997; 272: 25737–25742.
- ↵Barbulescu K, Becker C, Schlaak JF, Schmitt E, Meyer zum Buschenfelde KH, Neurath MF. IL-12 and IL-18 differentially regulate the transcriptional activity of the human IFN-γ promoter in primary CD4+T lymphocytes. J Immunol. 1998; 160: 3642–3647.
- ↵Mallat Z, Corbaz A, Scoazec A, Besnard S, Leseche G, Chvatchko Y, Tedgui A. Expression of interleukin-18 in human atherosclerotic plaques and relation to plaque instability. Circulation. 2001; 104: 1598–1603.
- ↵Piedrahita JA, Zhang SH, Hagaman JR, Oliver PM, Maeda N. Generation of mice carrying a mutant apolipoprotein-E gene inactivated by gene targeting in embryonic stem cells. Proc Natl Acad Sci U S A. 1992; 89: 4471–4475.
- ↵Dalton DK, Pitts-Meek S, Keshav S, Figari IS, Bradley A, Stewart TA. Multiple defects of immune cell function in mice with disrupted interferon-γ genes. Science. 1993; 259: 1739–1742.
- ↵Carson WE, Dierksheide JE, Jabbour S, Anghelina M, Bouchard P, Ku G, Yu H, Baumann H, Shah MH, Cooper MA, Durbin J, Caligiuri MA. Coadministration of interleukin-18 and interleukin-12 induces a fatal inflammatory response in mice: critical role of natural killer cell interferon-γ production and STAT-mediated signal transduction. Blood. 2000; 96: 1465–1473.
- ↵Kawakami K, Koguchi Y, Qureshi MH, Miyazato A, Yara S, Kinjo Y, Iwakura Y, Takeda K, Akira S, Kurimoto M, Saito A. IL-18 contributes to host resistance against infection with Cryptococcus neoformans in mice with defective IL-12 synthesis through induction of IFN-γ production by NK cells. J Immunol. 2000; 165: 941–947.
- ↵Micallef MJ, Ohtsuki T, Kohno K, Tanabe F, Ushio S, Namba M, Tanimoto T, Torigoe K, Fujii M, Ikeda M, Fukuda S, Kurimoto M, Interferon-γ–inducing factor enhances T helper 1 cytokine production by stimulated human T cells: synergism with interleukin-12 for interferon-γ production. Eur J Immunol. 1996; 26: 1647–1651.
- ↵Kohno K, Kataoka J, Ohtsuki T, Suemoto Y, Okamoto I, Usui M, Ikeda M, Kurimoto M. IFN-γ–inducing factor (IGIF) is a co-stimulatory factor on the activation of Th1 but not Th2 cells and exerts its effect independently of IL-12. J Immunol. 1997; 158: 1541–1550.
- ↵Zhang T, Kawakami K, Qureshi MH, Okamura H, Kurimoto M, Saito A. Interleukin-12 (IL-12) and IL-18 synergistically induce the fungicidal activity of murine peritoneal exudate cells against Cryptococcus neoformans through production of γ interferon by natural killer cells. Infect Immun. 1997; 65: 3594–3599.
- ↵Munder M, Mallo M, Eichmann K, Modolell M. Murine macrophages secrete interferon γ upon combined stimulation with interleukin (IL)-12 and IL-18: a novel pathway of autocrine macrophage activation. J Exp Med. 1998; 187: 2103–2108.
- ↵Geng YJ, Holm J, Nygren S, Bruzelius M, Stemme S, Hansson GK. Expression of the macrophage scavenger receptor in atheroma: relationship to immune activation and the T-cell cytokine interferon-γ. Arterioscler Thromb Vasc Biol. 1995; 15: 1995–2002.
- ↵Conrad DJ, Kuhn H, Mulkins M, Highland E, Sigal E. Specific inflammatory cytokines regulate the expression of human monocyte 15-lipoxygenase. Proc Natl Acad Sci U S A. 1992; 89: 217–221.
- ↵Jonasson L, Bondjers G, Hansson GK. Lipoprotein lipase in atherosclerosis: its presence in smooth muscle cells and absence from macrophages. J Lipid Res. 1987; 28: 437–445.
- ↵Fong LG, Fong TAT, Cooper AD. Inhibition of mouse macrophage degradation of acetyl low density lipoprotein by interferon-γ. J Biol Chem. 1990; 265: 11751–11760.
- ↵Geng YJ, Wu Q, Muszynski M, Hansson GK, Libby P. Apoptosis of vascular smooth muscle cells induced by in vitro stimulation with interferon-γ, tumor necrosis factor-α, and interleukin-1β. Arterioscler Thromb Vasc Biol. 1996; 16: 19–27.
- ↵Panousis CG, Zuckerman SH. Interferon-γ induces downregulation of Tangier disease gene (ATP-binding-cassette transporter 1) in macrophage-derived foam cells. Arterioscler Thromb Vasc Biol. 2000; 20: 1565–1571.