Influence of Interferon-γ on the Extent and Phenotype of Diet-Induced Atherosclerosis in the LDLR-Deficient Mouse
Objective— The aim of this study was to investigate the influence of interferon-γ (IFN-γ) on atherosclerosis in low density lipoprotein receptor (LDLR)–null mice.
Methods and Results— We cross-bred IFN-γ–deficient mice with LDLR-null mice and analyzed lipoprotein profiles and atherosclerosis in the compound mutant progeny after 8 and 20 weeks on a cholesterol-enriched diet. IFN-γ deficiency did not affect serum cholesterol levels or lipoprotein profiles, but it did affect the extent and phenotype of atherosclerosis. Atherosclerotic lesions in IFN-γ–deficient mice were reduced by 75% in the aortic arch and by 46% in the descending aorta compared with control mice after 8 weeks on the diet. After 20 weeks, arch lesions were reduced by 43%, and descending aorta lesions were reduced by 65% in IFN-γ–deficient mice compared with controls. At 8 weeks, percent lesional macrophage and smooth muscle content was significantly less in the IFN-γ–deficient mice, but not at 20 weeks. Although there were fewer class II major histocompatibility complex–positive cells in the lesions of IFN-γ–deficient animals compared with controls, class II major histocompatibility complex expression on endothelial cells overlying lesions persisted in the absence of IFN-γ.
Conclusions— These data provide direct evidence that IFN-γ influences atherosclerosis development and phenotype in the LDLR-deficient mouse, independent of changes in blood lipoprotein profiles.
Atherosclerotic lesions are sites of chronic inflammation in which cells and soluble mediators of both the innate and adaptive immune system are prevalent.1 CD4+ T lymphocytes are abundant in atheromata, and many of these T cells express markers of recent activation, such as the α-chain of the interleukin (IL)-2 receptor and major histocompatibility complex (MHC) class II molecules.2,3⇓ Immunohistochemical and in situ hybridization studies indicate that interferon-γ (IFN-γ) is often present in human atherosclerotic plaques,4–6⇓⇓ while Th2 cytokines, such as IL-4, are rarely detectable. Th1-mediated immune responses correlate with the promotion of atherosclerosis in mouse models.7,8⇓ These observations have led to the hypothesis that effector CD4+ T cells of the Th1 phenotype migrate into atherosclerotic lesions, become activated by lesional antigens, and secrete IFN-γ9,10⇓ and that IFN-γ exhibits proatherogenic effects.11–13⇓⇓
Genetically modified mice that are susceptible to atherosclerosis provide the opportunity to analyze the influence of cytokines, such as IFN-γ, on atherosclerosis.14–16⇓⇓ The published evidence in favor of a proatherogenic role of IFN-γ is limited to studies with apolipoprotein (Apo) E-null mice,17–19⇓⇓ but the results of these studies are inconsistent in several respects. Female IFN-γ receptor (IFN-γR)–deficient ApoE-null mice on a mixed genetic background were reported to develop less atherosclerosis than control ApoE-null mice, but the lipoprotein profiles differed between the experimental groups, with elevated ApoA-IV expression in the absence of IFN-γR.17 A second study showed that exogenously administered IFN-γ enhances atherosclerosis in ApoE-null mice and paradoxically reduces serum total and LDL cholesterol levels.18 In another study, IFN-γ deficiency in male but not in female ApoE-null mice was associated with reduced atherosclerosis, without changes in plasma lipoproteins.19 In consideration of these confusing results from ApoE-null mice and because the influences of IFN-γ on atherosclerosis in LDLR-null mice are largely unknown, we studied atherosclerosis and lipoprotein profiles in Ldlr−/−mice with or without a functional IFN-γ gene.
IFN-γ−/− mice and Ldlr−/− mice, backcrossed 10 times onto a C57BL/6 background, were purchased from Jackson Laboratories (Bar Harbor, Me). The 2 single-knockout strains were cross-bred, and the doubly heterozygous progeny were intercrossed to generate double-knockout IFN-γ−/−/Ldlr−/− mice and IFN-γ+/+/Ldlr−/− littermate controls. Animal genotype was identified by a polymerase chain reaction–based assay, as described previously.20,21⇓ All mice were housed and bred in accordance with the institutional guidelines of Brigham and Women’s Hospital and Harvard Medical School.
At 5 to 6 weeks of age, 21 IFN-γ−/−/Ldlr−/− or sex-matched IFN-γ+/+/Ldlr−/− (control) mice were recruited for this study and fed a semipurified cholate-free diet (No. D12108; Research Diets Inc) containing 40% kcal lipid and 1.25% cholesterol,14 ad libitum. After 8 or 20 weeks on this diet (n=11: 5 males and 6 females per group for the 8-week study; n=10: 5 males and 5 females per group for the 20-week study), the mice were fasted overnight and sacrificed by halothane inhalation. Blood was collected by vena cava nicking, and the arterial tree was perfused with Dulbecco’s phosphate-buffered saline (Gibco BRL). Perfused aortas were dissected from the aortic valve to the iliac bifurcation: the aortic arches were cut and separated from the remaining aorta and then rapidly frozen in optimal cutting temperature embedding medium (OCT, Tissue-Tek); the remaining thoracic and abdominal aorta (descending aorta) from each mouse was fixed in 10% buffered formalin.
Aortic Atherosclerotic Lesion Analysis
Two types of analyses were performed to quantify atherosclerotic lesions in the aortic arch or the descending aorta, respectively. First, longitudinal 5-μm cryostat sections of the aortic arch, 3 per specimen, were stained with oil red O (ORO).22 A defined portion of aortic arch wall was analyzed microscopically in all sections from each mouse, as described.23 This portion included a 2-mm segment of the lesser curvature, defined proximally by the aortic root and distally by a perpendicular axis from the distal side of the innominate artery origin. Medial, intimal, and lipid-positive areas, subtended by this 2-mm stretch of intima, were calculated for each mouse by computerized image analysis by using Imagepro Plus software (Media Cybernetics). Second, the remaining formalin-fixed thoracic and abdominal aorta from each mouse was stained with ORO, opened longitudinally, pinned out, and photographed with a digital camera to obtain images of lesions en face, as previously described.11 The percent surface area occupied by ORO-stained lesions viewed en face was determined by using Imagepro Plus software.
Immunohistochemistry, Immunofluorescence, and Histochemistry
For immunohistochemical analysis, serial longitudinal cryostat sections of aortic arch adjacent to the ORO-stained sections were stained, as described,23 with the respective cell-specific and isotype control antibodies, all from BD Pharmingen unless otherwise specified. Mouse-specific antibodies included the following: anti-CD31 (PECAM-1; clone MEC 13.3) for endothelial cells; anti-BM8 (Bachem) or anti-Mac3 (clone M3/84) for macrophages; anti–I-A/I-E (clone M5/114.15.2) for class II MHC; anti-CD4 (clone RM4-5) for CD4+ T cells; and anti–α-smooth muscle actin (clone 1A4, Sigma) for smooth muscle cells.
Endothelium and class II MHC–positive cells in atherosclerotic lesions were also stained with fluoroscein isothiocyanate (FITC) –anti mouse CD31 (PECAM-1; clone MEC 13.3) and phycoerythrin (PE)–anti-mouse I-Ab (clone AF6-120.I), respectively. For specificity controls, polyclonal FITC- or PE-labeled IgG2a,κ was used. The fluorescent staining was visualized by confocal microscopy. Collagen types I and III were stained by Picrosirius red, as described,24 and the sections were analyzed by polarization microscopy.
Serum Lipid Analysis
Serum lipoprotein profiles were determined by Superose 6 gel filtration fast-performance liquid chromatography (FPLC) from 7 animals (4 males, 3 females) in each group. Serum levels were expressed in mmol/L. To evaluate possible changes in serum lipoproteins, including ApoA-IV in more detail, serum samples were taken after 8 weeks of cholesterol-diet feeding from 4 IFN-γ−/−/ Ldlr−/− or sex-matched IFN-γ+/+/Ldlr−/−mice, and lipoproteins were fractionated sequentially by buoyant density ultracentrifugation at densities of 1.006, 1.063, and 1.21 g/mL and analyzed by SDS–polyacrylamide gel electrophoresis (PAGE).
Blood Leukocyte Counts
Total blood leukocytes were counted microscopically, and differential count was determined by multiparameter flow cytometry with lineage-specific antibodies (BD Pharmingen) including the following: PE–anti–Mac-1 (clone M1/70), FITC–anti–Gr-1(RB6-8C5), CyChrome-anti–CD45R-B220 (clone RA3-6B2), PE–anti-CD19 (clone ID3), and FITC–anti-CD3ε (145-2C11), as described.25
In Vitro Assays of CD4+ Cytokine Secretion
Spleen and lymph nodes were removed from 6 IFN-γ−/−/Ldlr−/− or IFN-γ+/+/Ldlr−/−mice after 8 weeks of the diet, and CD4+ T cells were isolated by anti-CD4 magnetic beads (Dynal). The cells were stimulated in microwell cultures (5×105/well) with plate-bound anti-CD3ε26 or with recombinant murine HSP60 (10 μg/mL), produced as described,27 plus syngeneic mitomycin C–treated spleen cells (5×106/well). Ovalbumin (10 μg/mL) and medium alone were used as controls. Culture supernatants were removed at 48 hours and analyzed by ELISA for IFN-γ, IL-4, and IL-10 cytokines with reagents from BD Pharmingen.
All statistical analyses were performed using Prism software. Differences between IFN-γ−/−/Ldlr−/− and IFN-γ+/+/Ldlr−/− mice for normally distributed data (all except aortic lipid and intimal area analyses) were analyzed by the Student’s t test and expressed as mean±SEM. The aortic lipid and intimal area data were analyzed by the Mann-Whitney U test. P≤0.05 was considered significant for all analyses.
Generation of IFN-γ−/−/Ldlr−/− Mice
Mice with homozygous null mutations in both the IFN-γ and Ldlr genes and littermate controls homozygous for the mutant Ldlr gene and the wild-type IFN-γ gene were generated and screened as described in Methods. There were no discernible differences in litter size or appearance of the IFN-γ–deficient versus control mice. There was also no significant difference in the weights of IFN-γ−/−/Ldlr−/− and IFN-γ+/+/Ldlr−/− mice before or after cholesterol-diet feeding.
Serum Cholesterol Lipoprotein Profiles
The analysis of mean total, VLDL, LDL, and HDL serum cholesterol levels, as determined by FPLC, revealed no statistical differences between IFN-γ−/−/Ldlr−/− and IFN-γ+/+/Ldlr−/− mice (Table 1), and there were no differences in lipoprotein profiles (please see http://atvb.ahajournals.org.). Of note, the distribution of apoproteins, as determined by SDS-PAGE, appeared the same in the 2 groups of mice (Figure 1), with no evidence of differences in ApoA-IV levels. There were no significant differences in lipid profiles when males and females were compared (not shown).
Blood Leukocyte Counts
There were no statistical differences in total blood leukocyte counts between IFN-γ–deficient and control mice: 5.0±0.8 versus 5.4±0.7 cells ×106/mL, P=0.24, respectively (data not shown). The monocyte, neutrophil, and B-lymphocyte fractions were not statistically different between IFN-γ–deficient and control mice. There were proportionately more T cells in the blood of IFN-γ–null mice compared with controls, 26.9±0.9 versus 16.2±2.7%, respectively, P=0.03.
Quantitative Analysis of Atherosclerotic Lesions
Atherosclerosis in both the aortic arch and descending aorta was reduced in IFN-γ–deficient mice compared with controls after 8 weeks of cholesterol-diet feeding (please see http://atvb.ahajournals.org.). Specifically, as shown in Figure 2, the median lipid-positive area in the segment of aortic arch analyzed in IFN-γ−/−/Ldlr−/− mice was 25% of the area in IFN-γ+/+/Ldlr−/− mice (0.005 vs 0.020 mm2, P=0.005), the median intimal area in IFN-γ−/−/Ldlr−/− mice was 38% of the area in IFN-γ+/+/Ldlr−/− mice (0.031 vs 0.081 mm2, P=0.0007), and the median medial area was slightly less in IFN-γ–deficient mice than in controls (0.096 vs 0.123 mm2, P=0.02; data not shown). The median en face aortic lesional area in the descending aorta was also significantly less in IFN-γ–deficient mice than in controls (2.8% vs 5.2%, P=0.0007). Analysis by sex confirmed less atherosclerosis in both IFN-γ−/− male mice versus control males (0.008 vs 0.031 mm2 in the aortic arches, P=0.05; 3.1% vs 6.3% total area in descending aortas, P=0.02) and IFN-γ−/− females versus control females (0.009 vs 0.031 mm2 in the aortic arches, P=0.009; 3.0% vs 4.8% total area in descending aortas, P=0.05; data not shown).
Aortic arch and descending aorta atherosclerosis was also reduced in IFN-γ–deficient mice compared with controls after 20 weeks on the cholesterol-enriched diet. There was progression of atherosclerotic disease in both IFN-γ–deficient and control groups at this later time point (Figure 2), but quantitative analysis revealed persistent differences between the 2 groups. Specifically, in aortic arch sections, the median lipid-positive area was still significantly less in IFN-γ–deficient mice than in controls (0.012 vs 0.021 mm2, P=0.005). In both groups, the intimal area in the arch sections was greater at 20 weeks than at 8 weeks, and there were no statistical differences between the 2 groups. The median en face lesional area in the descending aorta was greater at 20 weeks than at 8 weeks in both groups, but the increase was proportionately greater in mice with IFN-γ (≈3-fold) compared with mice without IFN-γ (≈2-fold). The median en face lesional area in the descending aorta of IFN-γ–deficient mice was 30% of the area in control mice (5.2% vs 17.1%, P=0.0001). Analysis by sex again confirmed less atherosclerosis in both IFN-γ−/− male mice versus control males (0.011 vs 0.021 mm2 in the aortic arches, P=0.05; 6.5% vs 25.6% total area in descending aortas, P=0.03) and IFN-γ−/− females versus control females (0.014 vs 0.022 mm2 in the aortic arches, P=0.03; 6.7% vs 14.2% total area in descending aortas, P=0.01; data not shown).
Phenotypic Analysis of Atherosclerotic Lesions
Analysis of the cellular content of atherosclerotic lesions in both groups of mice was determined by specific immunostaining for macrophages, CD4+ T cells, class II MHC–positive cells, and smooth muscle cells in aortic arch sections (Figure 3). Positively stained areas were quantified by image analysis and expressed as percentage of intimal area to normalize for overall differences in the size of lesions between the IFN-γ−/−/Ldlr−/− and control IFN-γ+/+/Ldlr−/− mice (Table 2).
After 8 weeks of the proatherogenic diet, there were no observable differences between IFN-γ–deficient versus control animals in intimal content of CD4+ cells. In contrast, smooth muscle cell content was significantly lower in aortic arch intimas of IFN-γ–deficient animals compared with control animals. Macrophage content was assessed with anti–Mac-3, which not only detected macrophages in the neointima but also stained cells in the media. We also used anti-BM8, which stained fewer cells but appeared more specific for intimal macrophages. Both Mac-3 and BM8 stainings were significantly lower in intimas from IFN-γ–deficient mice than control mice. Because IFN-γ is known to induce class II MHC expression on a variety of cell types, we examined the expression of class II MHC in the intimas of aortic arch sections. Predictably, we found significantly less class II MHC expression in the sections from IFN-γ–deficient mice compared with controls. Interestingly, MHC class II was present on endothelial cells overlying intimal lesions, even in the IFN-γ–deficient mice; this was confirmed by immunofluorescent staining with a different anti–class II MHC antibody (not shown). The early lesions examined after 8 weeks of the proatherogenic diet were generally collagen poor; there was no type I or type III collagen detected in the lesions in the IFN-γ–deficient mice, whereas some type I and III collagens were detected in lesions from only 4 of 11 control mice.
Although the IFN-γ–deficient mice continued to have significantly less atherosclerosis than control mice after 20 weeks of cholesterol-enriched diet feeding, the differences in the phenotype of the lesions were less pronounced at the later time point. No significant differences were found in the percentages of lesional area staining positive for macrophages, smooth muscle cells, or type I and III collagen content of aortic arch lesions at 20 weeks, although the absolute content of these constituents was less, given the smaller lesion sizes. Class II MHC expression remained lower in the absence of IFN-γ at 20 weeks.
In Vitro Assays of CD4+ Cytokine Secretion
CD4+ T cells isolated from diet-fed IFN-γ+/+/Ldlr−/− mice produced significant amounts of IFN-γ on restimulation with anti-CD3 or HSP-60, and as expected, T cells from IFN-γ−/−/ Ldlr−/− did not. Low levels of IL-4 and IL-10 were produced by the T cells from both groups, without significant differences (Table 3). Interestingly, CD4+ T cells from cholesterol-fed IFN-γ+/+/Ldlr−/− mice produced IFN-γ, but no IL-4 and IL-10, in response to the plaque antigen HSP-60. HSP-60 did not stimulate IFN-γ production, as predicted, nor IL-4 or IL-10 from the IFN-γ−/−T cells.
Several studies have demonstrated colocalization of T cells and a principal cytokine that they secrete, IFN-γ, in atherosclerotic lesions.4,6⇓ Evidence of a role for IFN-γ in atherogenesis is also provided from 3 ApoE-null mouse model studies, but several inconsistencies in the findings of those studies make interpretations difficult. Although IFN-γ receptor deficiency was reported to be associated with a reduction in atherosclerotic lesion size in female ApoE-null mice,17 IFN-γ deficiency was reported to be associated with reduced atherosclerosis only in male and not female ApoE-null mice.19 IFN-γ receptor deficiency was also associated with an increase in a distinct population of lipoprotein particles that are rich in ApoA-IV, phospholipid, and free cholesterol.17 However, the ApoA-IV–containing particles identified in that study are not typical of the usual HDL association of ApoA-IV reported in other mouse studies,28,29⇓ and no differences in serum lipoprotein profiles were seen in IFN-γ–deficient ApoE-null mice.19 Another study showed that in ApoE-null mice, administration of exogenous IFN-γ significantly increased lesion size and the number of T lymphocytes and class II MHC positive cells in lesions but paradoxically reduced serum total and LDL cholesterol levels.18 The studies presented here help to resolve some of the uncertainties about the proatherogenic effects of IFN-γ by analyzing the influence of IFN-γ on diet-induced atherosclerosis in the setting of LDLR deficiency. Our data indicate that IFN-γ enhances diet-induced atherosclerosis in LDLR-null mice without concomitant changes in lipoprotein profiles.
Early recruitment of lymphocytes to atherosclerotic lesions has been observed in the LDLR-deficient mice, but further development of lesions ensues without a proportional increase in T lymphocytes.30 For this reason, we first characterized early lesions in mice fed a proatherogenic diet for 8 weeks. We found that a lack of IFN-γ results in a significant decrease in early atherosclerotic lesion development. Specifically, the absence of IFN-γ was associated with a 75% decrease in lesion area in the aortic arch sections and a 46% decrease in the descending aortas. Although our study was not designed to independently test the effects of IFN-γ deficiency in males versus females, we still found significant differences in lesion size when we compared IFN-γ−/− males versus control males and IFN-γ−/− females versus control females. Intimal smooth muscle cells and macrophages were also decreased in the absence of IFN-γ. Interestingly, the decrease in atherosclerosis seen in the absence of IFN-γ was not associated with a decrease in CD4+ T cells normalized to intimal area of the lesions. Thus, the consequences of IFN-γ deficiency are not indirectly due to a lack of T-cell recruitment to lesions. It is also possible that additional sources of IFN-γ, besides T cells, may exist in atherosclerotic lesions, such as macrophages31 or smooth muscle cells.32 Thus, IFN-γ deficiency may limit the atherogenic influence of these other cell types.
Atherosclerosis was also reduced in the absence of IFN-γ after 20 weeks of cholesterol-enriched diet feeding. There was a 43% reduction in intimal lipid in the aortic arch and a 65% reduction in the lipid-staining area of the descending aorta. However, there was no significant reduction in intimal macrophages or smooth muscle cells, normalized for intimal area, in IFN-γ–deficient mice at 20 weeks, as there was at 8 weeks. It appears that IFN-γ influences both the cellular content and lipid accumulation in early lesions as well as continued lesion formation and lipid accumulation through 20 weeks. However, IFN-γ–independent mechanisms allow cellular migration and/or proliferation to proceed after 8 weeks.
Aberrant class II MHC expression has often been evoked as evidence for the presence of IFN-γ in atherosclerotic lesions, and by inference, the presence of activated T cells.3,12⇓ The data presented here show that arterial wall class II MHC expression is in fact diminished in the absence of IFN-γ; this could be attributable to decreased expression of class II MHC on intimal cells, as well as decreased numbers of intimal cells, such as macrophages, especially in 8-week lesions. Interestingly, even in the absence of IFN-γ, there still were some class II MHC–positive macrophages and significant numbers of class II MHC–expressing endothelial cells overlying the small lesions. Nonlesional endothelium was class II MHC–negative. These observations indicate that there is an IFN-γ–independent mechanism of inducing endothelial class II MHC expression overlying atherosclerotic lesions. This may be significant, because lesional endothelial cells could present plaque neoantigens to CD4+ T cells in early lesions, even before IFN-γ is produced.
In evaluating systemic immune responses, we have found that CD4+ T cells from cholesterol-fed IFN-γ+/+/Ldlr−/− mice produce IFN-γ but not IL-4 or IL-10 in response to HSP-60, a known lesional antigen implicated in the immunopathogenesis of atherosclerosis.33 The fact that neither HSP-60 nor anti-CD3 stimulated IL-4 or IL-10 production by CD4+ T cells from cholesterol-fed IFN-γ−/−/Ldlr−/− mice indicates that a compensatory Th2-like response did not occur in the absence of IFN-γ.
In contrast with previous studies in ApoE-null mice,17,18⇓ we did not find that IFN-γ deficiency resulted in significant alterations of serum cholesterol levels or cholesterol distribution between VLDL, LDL, and HDL. A previous report that IFN-γ receptor deficiency resulted in increased serum ApoA-IV in ApoE-null mice does not appear to be relevant to the effects of IFN-γ deficiency in this study. It is therefore very unlikely that the influence of IFN-γ on atherosclerosis that we observed was secondary to systemic changes in lipid metabolism.
In summary, cholesterol diet–induced atherosclerosis in LDLR-deficient mice is significantly reduced in the absence of IFN-γ. This study supports the concept that blockade of the effects of IFN-γ–producing T cells in the vessel wall is a potentially useful strategy for the control of atherosclerotic disease.
This work was supported by NIH grants HL56985 (to A.H. Lichtman and C. Buono) and HL48743 (to A.H. Lichtman) and by a grant from Peptor, LTD, Rehovot, Israel (to A.H. Lichtman).
Received September 10, 2002; revision accepted December 18, 2002.
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- ↵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.
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