JOURNAL OF INTERFERON AND CYTOKINE RESEARCH 22:661–670 (2002)© Mary Ann Liebert, Inc.

IFN-g Deficiency Exerts Gender-Specific Effects onAtherogenesis in Apolipoprotein E2/2 Mice

STEWART C. WHITMAN,1,2 PUNNAIVANAM RAVISANKAR,1 and ALAN DAUGHERTY1

ABSTRACT

We have shown recently that administration of exogenous interferon-g (IFN-g) to apolipoprotein E (apoE)2/2

mice augmented atherogenesis. In the present study, we examined whether deficiency of endogenous IFN-gwould reduce atherosclerosis in apoE2/2 mice. Compound-deficient mice were generated by crossing strain-matched IFN-g2/2 and apoE2/2 mice and comparing them to apoE2/2 mice. Groups of both genders werefed either a normal or a high-fat diet. IFN-g deficiency did not affect serum cholesterol concentrations orlipoprotein-cholesterol distributions in any groups. IFN-g deficiency had no effect on serum triglyceride con-centrations, except for an increase noted in males fed a normal diet. The extent of atherosclerosis was deter-mined in tissue sections of the ascending aorta and on the surface of the aortic arch. During feeding of nor-mal diets, IFN-g deficiency had no effect on the extent of atherosclerosis in female mice in either vascularbed. In contrast, in male mice fed normal diet, IFN-g deficiency markedly decreased lesion size in both vas-cular beds. During feeding of high-fat diets, IFN-g deficiency also had no effect on lesion size in females butprofoundly decreased lesion size in the aortic root of male mice. IFN-g deficiency had no effect on the abun-dance of T lymphocytes or MHC class II-positive cells in aortic root lesions of females. By comparison, boththese parameters were reduced in lesions of male mice. Therefore, IFN-g deficiency decreased atherogenesis,potentially by decreasing T lymphocyte presence and cell activation, without influencing serum cholesterolconcentrations. However, this effect is strikingly restricted to male mice.

661

INTRODUCTION

T LYMPHOCYTES HAVE BEEN IDENTIFIED in both human andmouse atherosclerotic lesions at all stages of lesion devel-

opment.(1) These cells express MHC class II and very late ac-tivation antigen-1 (VLA-1), which are consistent with activa-tion that would lead to elaboration of cytokines.(2) A role of Tlymphocytes in the development of atherosclerotic lesions isimplied from animal studies in which pharmacologic and ge-netic approaches to immune suppression have modified the de-velopment of atherosclerotic lesions.(3–6)

Interferon-g (IFN-g) is a major component of the acquiredimmune response and is a prominent cytokine secreted by ac-tivated T lymphocytes,(7) natural killer (NK) cells,(8) and mac-rophages.(9) IFN-g mRNA has been detected in atheroscleroticlesions from both humans(10–12) and mice.(13,14) IFN-g proteinhas also been detected by immunocytochemical analysis of hu-man(10) and mouse lesions.(15,16) This cytokine has the poten-tial to influence the development of atherosclerotic lesions

through a number of mechanisms that have been demonstratedin cultured cells. These include effects on 15-lipoxygenase,(17)

lipoprotein metabolism,(18) lipoprotein modification,(19)

lipoprotein cell recognition,(11,20,21) apoptosis,(22) extracellularmatrix (ECM) synthesis,(23) and macrophage ABCA1 expres-sion.(24) These diverse effects of IFN-g on cultured cells ren-der it difficult to predict whether this cytokine would promoteor retard atherogenesis.

The current limited evidence is consistent with IFN-g exert-ing a proatherogenic role. First, exogenous delivery of the cy-tokine by parenteral administration increased lesion size inmice.(25,26) Further evidence of a potential proatherogenic ef-fect of IFN-g was provided by determining the effects of IFN-g receptor deficiency on the development of lesions in femaleapolipoprotein E2/2 (apoE2/2) mice.(27) Deficiency of this cy-tokine receptor decreased lesion development in the aortic rootand enriched the content of ECM. Deficiency of IFN-g recep-tors led to unexpected changes in plasma lipoprotein andapolipoprotein concentrations, and, therefore, it was difficult to

1Gill Heart Institute, Division of Cardiovascular Medicine, University of Kentucky, Lexington, KY 40536.2Present address: University of Ottawa Heart Institute, Ottawa, Ontario, Canada, K1Y 4W7.

determine if the effects on lesion formation were caused by pe-ripheral or local effects. Furthermore, recent findings in strain-matched mice demonstrate that IFN-g receptor deficiencies donot necessarily mimic the effects of IFN-g deficiency.(28) Thishas led to speculation that there may be alternative ligands forthe IFN-g receptor or more than one receptor class for this cy-tokine.(28)

To directly define the role of IFN-g in the development ofatherosclerotic lesions, we determined the effects of IFN-g de-ficiency on lesion development in two arterial regions in age-matched, strain-matched, and gender-matched apoE2/2 mice.Because diet has been shown previously to influence lesion de-velopment in lymphocyte-deficient apoE2 /2 mice, the effectsof IFN-g deficiency were determined during feeding of bothnormal and high-fat diets.(5,6) Furthermore, as gender can af-fect immune responses,(25,29) we determined the effects of IFN-g deficiency in both female and male mice. We demonstratethat deficiency of IFN-g had no effect on atherosclerosis in ei-ther vascular bed during feeding of normal or high-fat diets infemale apoE2/2 mice. However, IFN-g deficiency had a strik-ing effect in reducing the size of atherosclerotic lesions in maleapoE2/2 mice, without changing serum cholesterol concentra-tions. This effect was associated with a decreased abundanceof T lymphocytes and MHC class II-positive cells.

MATERIALS AND METHODS

Animals and diets

apoE2/2 and IFN-g2/2 mice, backcrossed 10 times to theC57BL/6 strain, were obtained from the Jackson Laboratory(Bar Harbor, ME). IFN-g2/2 mice were bred to apoE2/2 miceto generate compound-deficient mice. PCR, as described pre-viously,(30,31) was used to identify mice homozygous for bothapoE and IFN-g deficiency.

Mice were kept on a standard laboratory diet for 9 monthsor, at 8 weeks of age, were placed on a diet supplemented with0.15% (w/w) cholesterol and 20% (w/w) butterfat (high-fat diet)(TD 88137, Harlan Teklad, Madison, WI) for 3 months. Mice

were housed in a pathogen-free facility, and all procedures wereapproved by the University of Kentucky Institutional AnimalCare and Use Committee.

Blood collection

Blood samples at termination of the study were collectedfrom anesthetized mice by puncture of the right ventricle. Bloodwas allowed to clot, and serum was obtained by centrifugation.

Serum lipid and lipoprotein quantification

Serum cholesterol and triglyceride concentrations were de-termined using commercially available assays (Wako Bioprod-ucts, Richmond, VA) as described previously.(25,32) Serum cho-lesterol measurements were standardized to samples providedby the Core Laboratory for Clinical Studies at Washington Uni-versity. Lipoprotein-cholesterol and lipoprotein-triglyceridedistributions from individual serum samples (4–6 mice pergroup) were determined following resolution by size exclusionchromatography (Biologic Workstation, Bio-Rad, Hercules,CA) using a Superose 6 column (Pharmacia LKB Biotechnol-ogy, Uppsala, Sweden) as described previously.(25,32)

Tissue collection and lesion analysis

Mice were perfused with phosphate-buffered saline (PBS),and the hearts were separated from the aorta at the base, em-bedded in OCT, and frozen at 220°C. Aortic tissue was re-moved from the ascending aorta to the iliac bifurcation andplaced in fresh 4% paraformaldehyde overnight at ambient tem-perature.

The extent of atherosclerosis in the ascending aorta was de-termined as described previously(25,33,34) from Oil Red O-stainedserial sections, 8 mm thick and collected 80 mm apart, startingat the region where the aortic sinus becomes the ascending aorta.The cross-sectional area of all lesions in a section was measuredand summed to determine the total lesion area per section. Le-sion size was determined by calculating the mean area in foursections. En face measurements of the percentage of intimal sur-face area covered by atherosclerotic lesions were determined for

WHITMAN ET AL.662

TABLE 1. SERUM LIPID CONCENTRATIONS

Serum concentration (mg/dl)

Diet Gender IFN-g genotype n Total cholesterol Triglyceride

Normal M 1/1 11 218 6 13a 105 6 19diet 2/2 10 284 6 21 211 6 20*

F. 1/1 9 185 6 20 70 6 222/2 10 217 6 34 74 6 20**

Modified M 1/1 10 543 6 33 295 6 46dietb 2/2 10 592 6 44 348 6 41

F. 1/1 10 516 6 13 258 6 122/2 10 515 6 15 271 6 29

aMeans 6 SEM.bStandard laboratory mouse feed supplemented with 0.15% (wt/wt) cholesterol and 20% (wt/wt) butterfat (TD 88137, Harlan

Teklad).*p 5 0.01 vs. male ApoE2/2 3 IFN-g1/1 mice.**p 5 0.01 vs. male ApoE2/2 3 IFN-g2/2 mice.

the aortic arch, as described previously.(5,32) Measurements oflesion size were verified by two observers.

Cellular and ECM characterization of atherosclerotic lesions

Immunocytochemistry and ECM staining were performed onserial sections of the ascending aorta adjacent to those stainedwith Oil Red O, as described previously.(25,32) For immunocy-tochemistry, the following sera/monoclonal antibodies (mAb)were used: rabbit antisera to mouse macrophages (AI-AD31240, 1:3000 dilution) (Accurate Chemical and ScientificCorp., Westbury, NY), an antimouse Thy1.2 antibody (01011D,7 mg/ml), (PharMingen, San Diego, CA), and an antimouseMHC class II antibody (ALU0041, 1:5 dilution) (Biosource In-ternational, Camarillo, CA). Endogenous tissue peroxidase wasablated prior to immunostaining by exposing tissue to H2O2,and the extent of nonspecific staining was examined by stain-ing serial sections in the absence of primary antibody. Extra-cellular collagen and elastin were visualized with Gomoritrichrome and Verhoeff’s stains, respectively.

T lymphocytes and MHC class II-positive cells were quan-tified by manually counting the number of immunoreactivecells, as described previously.(25,33,35)

Statistics

Data analysis was performed using SigmaStat 2.03 software(SPSS Inc., Chicago, IL). Differences between experimentalgroups were evaluated using a one-way ANOVA with all pair-wise comparisons of the mean responses to the different treat-ment groups conducted using the Tukey test after assuring thatthe data complied with the constraints of parametric analyses.When the normality test failed while performing the one-wayANOVA, a one-way ANOVA on Ranks (Kruskal-Wallis test)was performed, and subsequent pairwise comparisons of themedian values for the treatment groups to a designated controlgroup were done using the Dunnett’s method. Values with p ,

0.05 were considered to be statistically significant. Values arerepresented as mean 6 standard error of mean (SEM).

RESULTS

Serum lipid and lipoproteins

IFN-g deficiency did not affect serum cholesterol concen-trations in any of the groups studied (Table 1). Lipoprotein-cho-lesterol distribution was also not influenced by IFN-g defi-ciency in apoE2 /2 mice of either gender fed normal or high-fat

IFN-g DEFICIENCY AND ATHEROSCLEROSIS 663

FIG. 1. Serum lipoprotein distribution of mice fed a normal and high-fat diet. Serum from either (A,C) male or (B,D) femaleapoE2/2 3 IFN-g1 /1 (open circle) or apoE2 /2 3 IFN-g2/2 (closed circle) mice fed a normal diet for 9 months (A,B) or a dietenriched in cholesterol and saturated fat for 3 months (C,D) was resolved by size exclusion chromatography using a Superose 6column, and cholesterol concentrations were determined in each fraction. Volumes of serum were loaded onto the column in ap-proximate proportion to the serum cholesterol concentrations (200 ml for normal diet and 50 ml for high-fat diet). Values are rep-resented as mean 6 SEM of individual profiles from 6–8 animals per group (A,B) and 4–6 animals per group (C,D).

diets (Fig. 1). IFN-g deficiency had no effect on serum triglyc-eride concentrations, except for the increase noted in apoE2 /2

male mice fed a normal diet (Table 1).

Quantification of atherosclerosis

In female apoE2/2 mice, IFN-g deficiency did not reducethe size of atherosclerotic lesions in either the aortic arch (Fig.2A,B) or the ascending aorta (Fig. 2C,D) during feeding of ei-ther normal or high-fat diets. Interestingly, lesions from the as-cending aorta in female mice were larger than in males (femalevs. male IFN-g2 /2 mice fed a normal diet, p 5 0.004 [Fig. 2C]and female vs. male IFN-g1/1 and IFN-g2 /2 mice fed a high-fat diet, p , 0.001 [Fig. 2D]).

In contrast to females, IFN-g deficiency reduced atheroscle-rosis in male apoE2/2 mice fed a normal diet for 9 months inboth the aortic arch (26.8% 6 2.5% vs. 14.4% 6 2.7%, p 5

0.007) (Fig. 2A) and ascending aorta (0.452 6 0.022 mm vs.0.262 6 0.038 mm2, p # 0.004) (Fig. 2C). In male apoE2 /2

mice fed a high-fat diet, although there was no significant ef-fect of IFN-g deficiency on the development of atherosclerosisin the aortic arch (Fig. 2B), there was a 74% decrease in lesionsize in the ascending aorta (0.255 6 0.036 mm2 vs. 0.067 6

0.008 mm2, p # 0.001) (Fig. 2D).

Characteristics of atherosclerotic lesions

Representative sections of atherosclerotic lesions from theascending aorta of male IFN-g1/1 and IFN-g2 /2 mice fed ei-

ther normal or high-fat diets are shown in Figures 3 and 4, re-spectively. Serial sections have been stained for neutral lipid(Figs. 3A,B and 4A,B), macrophages (Figs. 3C,D and 4C,D),T lymphocytes (Figs. 3E,F and 4E,F), and collagen (Figs. 3G,Hand 4G,H). Despite the reduction in the size of lesions in maleIFN-g2/2 3 apoE2 /2 mice, there were no obvious changes ingross morphology of lesions between the wild-type and cyto-kine-deficient mice. In all groups of mice, lesions were pre-dominantly composed of macrophages, many of which stainedfor neutral lipid (Figs. 3A,B,C,D and 4A,B,C,D). No gross dif-ferences in ECM elements of elastin or collagen were noted inlesions from apoE2 /2 male IFN-g1/1 vs. IFN-g2/2 mice, fol-lowing visualization with Verhoeff’s and Gomori stains (Figs.3G,H and 4G,H) (data not shown).

Quantification of cellularity of lesions

After visually detecting a difference in morphologic stainingpatterns for T lymphocytes, we conducted extensive quantita-tive analysis of both T lymphocyte-positive and MHC class II-positive cell populations within these lesions. No difference inlesion T lymphocyte numbers was found between apoE2/2 fe-male IFN-g1/1 and IFN-g2 /2 mice fed either diet (Fig. 5A,B).Comparison between genders revealed that lesions in femaleIFN-g2/2 mice had nearly twice as many T lymphocytes as le-sions from male IFN-g2 /2 mice fed a normal diet (p 5 0.037)(Fig. 5A). T lymphocyte abundance did not change in lesionsfrom apoE2/2 male IFN-g1/1 vs. IFN-g2/2 mice fed a normal

WHITMAN ET AL.664

FIG. 2. Aortic lesion size. The extent of intimal surface covered by grossly discernible lesions was determined from (A,B) enface preparations of the aortic arch and from (C,D) serial sections of the ascending aorta from male and female apoE2 /2 3 IFN-g1/1 (open symbols) and apoE2 /2 3 IFN-g2/2 (closed symbols) mice fed either (A,C) a normal diet for 9 months or (B,D) ahigh-fat diet for 3 months. Circles, values of individual mice; squares, means; bars, SEM.

IFN-g DEFICIENCY AND ATHEROSCLEROSIS 665

FIG. 3. Aortic lesion histology from mice fed a normal diet. Representative histologic sections from a region where the aorticsinus becomes the ascending aorta of a male (A,C,E,G) apoE2 /2 3 IFN-g1/1 mouse and a male (B,D,F,H) apoE2 /2 3 IFN-g2/2 mouse fed a normal diet for 9 months. A segment of heart tissue spanning the aortic sinus and ascending aorta was em-bedded in OCT, sectioned, and stained with (A,B) Oil Red O for neutral lipids, (C,D) rabbit antisera to mouse macrophages(1:3000 dilution), (E,F) a monoclonal antimouse Thy1.2 antibody (7 mg/ml), (G,H) Gomori trichrome to detect collagen. A,B, 340; C–H, 3200. (A, inset) and (B, inset) Areas contained in C,E,G and D,F,H, respectively.

diet (60 6 10 vs. 38 6 10 mean lesion cell number per section)(Fig. 5A) but was significantly decreased in mice fed a high-fat diet (50 6 8 vs. 15 6 5 mean lesion cell number per sec-tion, p 5 0.01) (Fig. 5B).

Substantial changes in the number of cells expressing MHCclass II antigens at the sites of inflammation are indicative ofthe intensity of an immune response. Therefore, we determinedwhether IFN-g deficiency affected MHC class II expression.IFN-g deficiency had no effect on the number of cells ex-pressing MHC class II in female apoE2/2 mice (Fig. 5C,D).MHC class II-expressing cells were significantly reduced inIFN-g2/2 3 apoE2 /2 male mice fed a normal diet (52 6 13 vs.13 6 3 cell number per section, p 5 0.004) (Fig. 5C) but notin lesions from mice fed a high-fat diet (24 6 6 vs. 9 6 4 meancell number per section) (Fig. 5D).

DISCUSSION

Previously, we have demonstrated that administration of ex-ogenous IFN-g significantly increased atherosclerosis inapoE2/2 male mice.(25) The present study provides further ev-idence that IFN-g is proatherogenic by demonstrating that en-dogenous deficiency of the cytokine in apoE2 /2 mice decreasesatherosclerosis. However, the effects of IFN-g deficiency onthe development of atherosclerosis are restricted to male mice.

Although both our study and that using IFN-g receptor-defi-cient mice(27) led to the same conclusion regarding the proathero-genic effects of this cytokine, there are several major differencesbetween the studies. Perhaps the most striking difference is thatIFN-g receptor deficiency reduced atherosclerosis in females,(27)

whereas in our study, deficiency of the cytokine had no effect onthis gender. The effects of IFN-g receptor deficiency on the de-velopment of atherosclerosis in male mice has not been reported.Also, in contrast to the receptor-deficient animals,(23) we werenot able to detect an effect of IFN-g deficiency on serum con-centrations of total cholesterol or lipoprotein-cholesterol distri-bution. Furthermore, IFN-g receptor-deficient animals had agrossly increased ECM content of lesions.(23) By comparison, wewere unable to detect any overt changes in the content of colla-gen and elastin in cytokine-deficient apoE2/2 mice.

As noted earlier, the differences in the atherogenic processin apoE2 /2 mice that lack either the cytokine or receptor maybe accounted for by a disparate phenotype of IFN-g and IFN-g receptor-deficient mice. The possibility of ligands other thanIFN-g interacting with the IFN-g receptor has been inferredpreviously in response to viral infection,(28) in a manner anal-ogous to the chemokine system.(36) However, there is currentlyno evidence that the difference is attributable to the existenceof more than one IFN-g receptor.(37) In addition to the poten-tial for differences between receptor and cytokine deficiency,the discrepancies between the two studies may also be attrib-utable to the different strains of mice. Gupta et al.(27) used ahybrid of 129 and C57BL/6 in both the single and compound-deficient mice. Both the apoE2 /2 and IFN-g2/2 mice used inthe present study were backcrossed 10 times into a C57BL/6background. In addition to ensuring strain equivalency in thesingle and compound-deficient mice, this strain would be oneof the most responsive to IFN-g deficiency, as C57BL/6 miceare considered among the most Th1-responsive strains.(30)

During the course of this study, we attempted to detect IFN-g protein in atherosclerotic lesions by immunocytochemicaltechniques. Previous studies have observed IFN-g in lesions bythis technique in human(10) and mouse tissue.(39) Also, IFN-gprotein has been detected in plasma(40) and spleens(13) of ath-erosclerotic animals, although its relationship to the local eventswithin vascular lesions is unclear. In agreement with Mallat etal.,(16) however, we were unable to detect IFN-g protein in ath-erosclerotic lesions from apoE2/2 mice. Part of the reason forthe inability to discern IFN-g may relate to the protein’s beingsecreted at intervals other than those at which the tissue wasacquired. In addition, very small amounts of IFN-g, which maybe below the limits of detection, exert pronounced biologic ef-fects through polarized delivery. Therefore, the inability to de-tect this cytokine does not diminish its role in the disease. Mostimportantly, in spite of our inability to detect IFN-g protein inthe lesions of mice that were wild-type for this cytokine, thedecrease in atherosclerosis in the IFN-g2 /2 mice demonstratesthat the cytokine is involved in the disease process.

Previous studies have demonstrated that there is a correla-tion between the extent of atherosclerosis in the aortic root andthroughout the entire aorta in apoE2/2 mice,(38) although dif-ferences have been noted.(41,42) Therefore, we chose to analyzelesion development in both these regions. For mice fed a nor-mal diet, we noted equivalent changes in both the aortic rootand the intimal area of the aortic arch. However, discrepanciesin effect of these two regions were noted in mice fed a high-fat diet. Under these conditions, IFN-g deficiency promotedpronounced decreases in lesion size in the aortic root but hadno effect on the en face analysis of the aortic intima. The rea-sons for disparities in the atherogenesis in different vascularbeds that have been described in this and other studies(37,39) isnot known.(42,43)

Atherosclerotic lesions of apoE2 /2 mice contain T lympho-cytes.(23,27,35,44) Therefore, lesions in these mice have the ap-propriate cellular constituents to study the role of this cell typein the disease process. Total lymphocyte deficiency decreasesatherosclerosis in apoE2 /2 mice, although this effect is not man-ifested during feeding of diets that promote a severe hyper-cho-lesterolemic response (serum cholesterol .1000 mg/dl).(5,6) Incontrast to the effects of high-fat diets ablating the role of lym-phocytes in atherogenesis in wild-type mice, IFN-g deficiencyreduced atherosclerosis when male mice were fed either nor-mal or high-fat diets. Future studies will address whether thisdifference is due to release of IFN-g from nonlymphocyticsources.(45)

Despite not observing any gross differences in overt cel-lular or ECM content between lesions from apoE2 /2 maleIFN-g2 /2 vs. IFN-g1 /1 mice, we did observe a significantreduction in the number of lesion-associated T lymphocytesand MHC class II-positive cells, suggesting that deficiencyof the cytokine reduced an inflammatory component of le-sion formation. IFN-g deficiency does not affect systemiclymphocyte populations,(31) suggesting that one of the ef-fects of IFN-g on the pathology is to enhance T lymphocytenumbers, via recruitment or clonal expansion, and the acti-vation of immunologic cells within the developing athero-sclerotic lesion. Another potential source of IFN-g may beNK cells that express this cytokine abundantly(8) and havebeen found in human and mouse atherosclerotic lesions.(46)

WHITMAN ET AL.666

IFN-g DEFICIENCY AND ATHEROSCLEROSIS 667

FIG. 4. Aortic lesion histology from mice fed a high-fat diet. Representative histologic sections from a region where the aor-tic sinus becomes the ascending aorta of a male (A,C,E,G) apoE2/2 3 IFN-g1/1 mouse and a male (B,D,F,H) apoE2 /2 3 IFN-g2/2 mouse fed a cholesterol and fat-enriched diet for 3 months. A segment of heart tissue spanning the aortic sinus and as-cending aorta was embedded in OCT, sectioned, and stained with (A,B) Oil Red O for neutral lipids, (C,D) rabbit antisera tomouse macrophages (1:3000 dilution), (E,F) a monoclonal antimouse Thy1.2 antibody (7 mg/ml), (G,H) Gomori trichrome todetect collagen. A,B, 340; C–H, 3200. (A, inset) and (B, inset) Areas contained in C,E,G and D,F,H, respectively.

At present, the contribution of this cell type to IFN-g se-cretion within lesions and progression of the disease is un-known.(46)

Female C56BL/6 mice develop more atherosclerosis thanmales,(47) but the data on gender differences have been moremixed in apoE2 /2 mice. Some have indicated an increasedextent of lesion formation in female mice.(40,48) However, thishas not been a uniformly observed difference and may dependon factors, such as age.(6,49) In the present study, the differ-ence was apparent only during the feeding of high-fat dietsand only in the ascending aorta. In agreement with the effectswe noted for IFN-g deficiency, gender-specific effects havealso been observed on the extent of atherosclerosis develop-ment in infections(50) and total lymphocyte deficiency.(6,42) Inboth cases, males exhibited an effect on atherosclerosis in re-sponse to the intervention, whereas females were unaffected.Gender-specific differences in the relative lymphocyte sub-type responses have been described in both humans and mice,with females exhibiting more pronounced Th2 responses com-pared with Th1-oriented response in males.(29,51) Although the

basis for this difference in responsiveness has not been de-fined, a hormonal basis is indicated by the switch in Th1:Th2responses that occurs during pregnancy.(52,53) Furthermore,these changes revert to normal in the postpartum phase.(54,55)

Therefore, it remains to be determined whether a specific hor-monal balance is responsible for lack of effect of IFN-g de-ficiency on the size and cellular characteristics of lesions infemale apoE2 /2 mice.

In summary, we found a striking difference between maleand female mice in the effect of IFN-g deficiency on athero-sclerosis. Deficiency of endogenous IFN-g decreased the de-velopment of atherosclerotic lesions in male mice. This de-crease was associated with a decreased abundance of Tlymphocytes and MHC class II-positive cells. Because the abil-ity to mount an effective immunologic response involves an in-tricate array of cytokines from various immunologic cells, morework must be done to determine the mediators underlying thisdifference. In future studies, we will determine the role of gen-der-specific hormones, particularly estrogen, on the effects ofIFN-g on the atherogenic process.(56)

WHITMAN ET AL.668

FIG. 5. Aortic lesion T lymphocyte and MHC class II-positive cell numbers. Numbers of lesion-associated (A,B) T lympho-cyte-positive and (C,D) MHC class II-positive cells were determined from cross-sections of the ascending aorta of male and fe-male apoE2/2 3 IFN-g2/2 (open symbols) and apoE2/2 3 IFN-g2 /2 (closed symbols) mice fed either (A,C) a normal diet for9 months or (B,D) a high-fat diet for 3 months. Circles, values of individual mice; squares, means; bars, SEM. Segments of hearttissue spanning the aortic sinus and ascending aorta were embedded in OCT, sectioned, and stained with (A,B) a monoclonal an-timouse Thy1.2 antibody (7 mm/ml) or (C,D) a monoclonal antimouse MHC II antibody (1:5 dilution). Values are derived from9 mice per group in A and C and from 6 mice per group in B and D.

ACKNOWLEDGMENTS

This work was supported by fellowships to S.C.W. from theHeart and Stroke Foundation of Canada and the American HeartAssociation (Ohio Valley Affiliate) and an American Heart As-sociation National Grant-in-Aid (A.D.).

REFERENCES

1. DAUGHERTY, A., and HANSSON, G.K. (2000). Lymphocytesin atherogenesis. In: R.T. Dean and D. Kelly (eds.) Atherosclero-sis. Oxford: Oxford University Press, pp. 230–249.

2. STEMME, S., HOLM, J., and HANSSON, G.K. (1992). T lym-phocytes in human atherosclerotic plaques are memory cells ex-pressing CD45RO and the integrin VLA-1. Arterioscler. Thromb.12, 206–211.

3. ROSELAAR, S.E., SCHONFELD, G., and DAUGHERTY, A.(1995). Enhanced development of atherosclerosis in cholesterol-fed rabbits by suppression of cell-mediated immunity. J. Clin. In-vest. 96, 1389–1394.

4. DREW, A.F., and TIPPING, P.G. (1995). Cyclosporine treatmentreduces early atherosclerosis in the cholesterol-fed rabbit. Athero-sclerosis 116, 181–189.

5. DAUGHERTY, A., PURE, E., DELFEL-BUTTEIGER, D.,CHEN, S., LEFEROVICH, J., ROSELAAR, S.E., and RADER,D.J. (1997). The effects of total lymphocyte deficiency on the ex-tent of atherosclerosis in apolipoprotein E2/2 mice. J. Clin. Invest.100, 1575–1580.

6. DANSKY, H.M., CHARLTON, S.A., HARPER, M.M., andSMITH, J.D. (1997). T and B lymphocytes play a minor role inatherosclerotic plaque formation in the apolipoprotein E-deficientmouse. Proc. Natl. Acad. Sci. USA 94, 4642–4646.

7. ABBAS, A.K., WILLIAMS, M.E., BURSTEIN, H.J., CHANG, T.-L., BOSSU, P., and LICHTMAN, A.H. (1991). Activation andfunctions of CD41 T-cell subsets. Immunol. Rev. 123, 5–22.

8. KIM, S., IIZUKA, K., AGUILA, H.L., WEISSMAN, I.L., andYOKOYAMA, W.M. (2000). In vivo natural killer cell activitiesrevealed by natural killer cell-deficient mice. Proc. Natl. Acad. Sci.USA 97, 2731–2736.

9. GESSANI, S., and BELARDELLI, F. (1998). IFN-gamma ex-pression in macrophages and its possible biological significance.Cytokine Growth Factor Rev. 9, 117–123.

10. HANSSON, G.K., HOLM, J., and JONASSON, L. (1989). Detec-tion of activated T lymphocytes in the human atheroscleroticplaque. Am. J. Pathol. 135, 169–175.

11. GENG, Y.J., HOLM, J., NYGREN, S., BRUZELIUS, M.,STEMME, S., and HANSSON, G.K. (1995). Expression of themacrophage scavenger receptor in atheroma—relationship to im-mune activation and the T-cell cytokine interferon-gamma. Arte-rioscler. Thromb. Vasc. Biol. 15, 1995–2002.

12. UYEMURA, K., DEMER, L.L., CASTLE, S.C., JULLIEN, D.,BERLINER, J.A., GATELY, M.K., WARRIER, R.R., PHAM, N.,FOGELMAN, A.M., and MODLIN, R.L. (1996). Cross-regulatoryroles of interleukin (IL)-12 and IL-10 in atherosclerosis. J. Clin.Invest. 97, 2130–2138.

13. ZHOU, X.H., PAULSSON, G., STEMME, S., and HANSSON,G.K. (1998). Hypercholesterolemia is associated with a T helper(Th1) 1/Th2 switch of the autoimmune response in atheroscleroticapo E-knockout mice. J. Clin. Invest. 101, 1717–1725.

14. LEE, T.S., YEN, H.C., PAN, C.C., and CHAU, L.Y. (1999). Therole of interleukin 12 in the development of atherosclerosis inApoE-deficient mice. Arterioscler. Thromb. Vasc. Biol. 19, 734–742.

15. HUBER, S.A., SAKKINEN, P., DAVID, C., NEWELL, M.K., and

TRACY, R.P. (2001). T helper-cell phenotype regulates athero-sclerosis in mice under conditions of mild hypercholesterolemia.Circulation 103, 2610–2616.

16. MALLAT, Z., BESNARD, S., DURIEZ, M., DELEUZE, V., EM-MANUEL, F., BUREAU, M.F., SOUBRIER, F., ESPOSITO, B.,DUEZ, H., FIEVET, C., STAELS, B., DUVERGER, N., SCHER-MAN, D., and TEDGUI, A. (1999). Protective role of interleukin-10 in atherosclerosis. Circ. Res. 85, E17–E24.

17. CONRAD, D.J., KUHN, H., MULKINS, M., HIGHLAND, E., andSIGAL, E. (1992). Specific inflammatory cytokines regulate theexpression of human monocyte 15-lipoxygenase. Proc. Natl. Acad.Sci. USA 89, 217–221.

18. JONASSON, L., BONDJERS, G., and HANSSON, G.K. (1987).Lipoprotein lipase in atherosclerosis: its presence in smooth mus-cle cells and absence from macrophages. J. Lipid Res. 28, 437–445.

19. CHRISTEN, S., THOMAS, S.R., GARNER, B., and STOCKER,R. (1994). Inhibition by interferon-gamma of human mononuclearcell-mediated low density lipoprotein oxidation—participation oftryptophan metabolism along the kynurenine pathway. J. Clin. In-vest. 93, 2149–2158.

20. FONG, L.G., FONG, T.A.T., and COOPER, A.D. (1990). Inhibi-tion of mouse macrophage degradation of acetyl low densitylipoprotein by interferon-gamma. J. Biol. Chem. 265, 11751–11760.

21. LAMARRE, J., WOLF, B.B., KITTLER, E.L.W., QUESEN-BERRY, P.J., and GONIAS, S.L. (1993). Regulation of macro-phage alpha2-macroglobulin receptor/low density lipoprotein re-ceptor-related protein by lipopolysaccharide and interferon-gamma. J. Clin. Invest. 91, 1219–1224.

22. GENG, Y.J., WU, Q., MUSZYNSKI, M., HANSSON, G.K., andLIBBY, P. (1996). Apoptosis of vascular smooth muscle cells in-duced by in vitro stimulation with interferon-gamma, tumor ne-crosis factor-alpha, and interleukin-1 beta. Arterioscler. Thromb.Vasc. Biol. 16, 19–27.

23. SEMPOWSKI, G.D., DERDAK, S., and PHIPPS, R.P. (1996). In-terleukin-4 and interferon-gamma discordantly regulate collagenbiosynthesis by functionally distinct lung fibroblast subsets. J. Cell.Physiol. 167, 290–296.

24. PANOUSIS, C.G., and ZUCKERMAN, S.H. (2000). Interferon-gamma induces downregulation of Tangier disease gene (ATP-binding-cassette transporter 1) in macrophage-derived foam cells.Arterioscler. Thromb. Vasc. Biol. 20, 1565–1571.

25. WHITMAN, S.C., RAVISANKAR, P., ELAM, H., and DAUGH-ERTY, A. (2000). Exogenous interferon-gamma enhances athero-sclerosis in apolipoprotein E2/2 mice. Am. J. Pathol. 157, 1819–1824.

26. TELLIDES, G., TEREB, D.A., KIRKILESSMITH, N.C., KIM,R.W., WILSON, J.H., SCHECHNER, J.S., LORBER, M.I., andPOBER, J.S. (2000). Interferon-gamma elicits arteriosclerosis inthe absence of leukocytes. Nature 403, 207–211.

27. GUPTA, S., PABLO, A.M., JIANG, X.C., WANG, N., TALL,A.R., and SCHINDLER, C. (1997). IFN-gamma potentiates ather-osclerosis in apoE knock-out mice. J. Clin. Invest. 99, 2752–2761.

28. CANTIN, E., TANAMACHI, B., OPENSHAW, H., MANN, J.,and CLARKE, K. (1999). Gamma interferon (IFN-gamma) recep-tor null-mutant mice are more susceptible to herpes simplex virustype 1 infection than IFN-gamma ligand null-mutant mice. J. Vi-rol. 73, 5196–5200.

29. HUBER, S.A., and PFAEFFLE, B. (1994). Differential Th1 andTh2 cell responses in male and female BALB/c mice infected withcoxsackievirus group B type 3. J. Virol. 68, 5126–5132.

30. PIEDRAHITA, J.A., ZHANG, S.H., HAGAMAN, J.R., OLIVER,P.M., and MAEDA, N. (1992). Generation of mice carrying a mu-tant apolipoprotein-E gene inactivated by gene targeting in em-bryonic stem cells. Proc. Natl. Acad. Sci. USA 89, 4471–4475.

31. DALTON, D.K., PITTS-MEEK, S., KESHAV, S., FIGARI, I.S.,

IFN-g DEFICIENCY AND ATHEROSCLEROSIS 669

BRADLEY, A., and STEWART, T.A. (1993). Multiple defects ofimmune cell function in mice with disrupted interferon-g genes.Science 259, 1739–1742.

32. DAUGHERTY, A., MANNING, M.W., and CASSIS, L.A. (2000).Angiotensin II promotes atherosclerotic lesions and aneurysms inapolipoprotein E-deficient mice. J. Clin. Invest. 105, 1605–1612.

33. WHITMAN, S.C., RAVISANKAR, P., and DAUGHERTY, A.(2002). Interleukin-18 enhances atherosclerosis in apolipoproteinE(2/2) mice through release of interferon-gamma. Circ. Res. 90,E34–38.

34. KING, V.L., SZILVASSY, S.J., and DAUGHTERY, A. (2002).Interleukin-4 deficiency decreases atherosclerotic lesion formationin a site-specific manner in female LDL receptor 2/2 mice. Arte-rioscler. Thromb. Vasc. Biol. 22, 456–461.

35. ROSELAAR, S.E., KAKKANATHU, P.X., and DAUGHERTY,A. (1996). Lymphocyte populations in atherosclerotic lesions ofapoE 2/2 and LDL receptor 2/2 mice. Decreasing density with dis-ease progression. Arterioscler. Thromb. Vasc. Biol. 16, 1013–1018.

36. REAPE, T.J., and GROOT, P.H.E. (1999). Chemokines and ath-erosclerosis. Atherosclerosis 147, 213–225.

37. STARK, G.R., KERR, I.M., WILLIAMS, B.R., SILVERMAN,R.H., and SCHREIBER, R.D. (1998). How cells respond to inter-ferons. Annu. Rev. Biochem. 67, 227–264.

38. TANGIRALA, R.K., RUBIN, E.M., and PALINSKI, W. (1995).Quantitation of atherosclerosis in murine models: correlation be-tween lesions in the aortic origin and in the entire aorta, and dif-ferences in the extent of lesions between sexes in LDL receptor-deficient and apolipoprotein E-deficient mice. J. Lipid Res. 36,2320–2328.

39. GEORGE, J., SCHOENFELD, Y., GILBURD, B., AFEK, A.,SHAISH, A., and HARATS, D. (2000). Requisite role for inter-leukin-4 in the acceleration of fatty streaks induced by heat shockprotein 65 or Mycobacterium tuberculosis. Circ. Res. 86, 1203–1210.

40. ZHOU, X.H., NICOLETTI, A., ELHAGE, R., and HANSSON,G.K. (2000). Transfer of CD4(1) T cell aggravates atherosclerosisin immunodeficient apolipoprotein E knockout mice. Circulation102, 2919–2922.

41. WITTING, P.K., PETTERSSON, K., LETTERS, J., andSTOCKER, R. (2000). Site-specific antiatherogenic effect ofprobucol in apolipoprotein E-deficient mice. Arterioscler. Thromb.Vasc. Biol. 20, E26–E33.

42. VENIANT, M.M., PIEROTTI, V., NEWLAND, D., CHAM, C.M.,SANAN, D.A., WALZEM, R.L., and YOUNG, S.G. (1997). Sus-ceptibility to atherosclerosis in mice expressing exclusivelyapolipoprotein B48 or apolipoprotein B100. J. Clin. Invest. 100,180–188.

43. ROSENFELD, M.E., POLINSKY, P., VIRMANI, R., KAUSER,K., RUBANYI, G., and SCHWARTZ, S.M. (2000). Advanced ath-erosclerotic lesions in the innominate artery of the ApoE knockoutmouse. Arterioscler. Thromb. Vasc. Biol. 20, 2587–2592.

44. ZHOU, X.H., STEMME, S., and HANSSON, G.K. (1996). Evi-dence for a local immune response in atherosclerosis: CD4(1) Tcells infiltrate lesions of apoliproprotein-E-deficient mice. Am. J.Pathol. 149, 359–366.

45. LOUIS, J., HIMMELRICH, H., PARRA-LOPEZ, C., TACCHINI-COTTIER, F., and LAUNOIS, P. (1998). Regulation of protective

immunity against Leishmania major in mice. Curr. Opin. Immunol.10, 459–464.

46. CURTISS, L.K., KUBO, N., SCHILLER, N.K., and BOISVERT,W.A. (2000). Participation of innate and acquired immunity in ath-erosclerosis. Immunol. Res. 21, 167–176.

47. PAIGEN, B., HOLMES, P.A., MITCHELL, D., and ALBEE, D.(1987). Comparison of atherosclerotic lesions and HDL-lipid lev-els in male, female, and testosterone-treated female mice fromstrains C57BL/6, BALB/c, and C3H. Atherosclerosis 64, 215–221.

48. SMITH, J.D., TROGAN, E., GINSBERG, M., GRIGAUX, C.,TIAN, J., and MIYATA, M. (1995). Decreased atherosclerosis inmice deficient in both macrophage colony-stimulating factor (op)and apoliproprotein E. Proc. Natl. Acad. Sci. USA 92, 8264–8268.

49. CALIGIURI, G., NICOLETTI, A., ZHOU, X.H., TORNBERG, I.,and HANSSON, G.K. (1999). Effects of sex and age on athero-sclerosis and autoimmunity in apoE-deficient mice. Atherosclero-sis 145, 301–308.

50. BURNETT, M.S., GAYDOS, C.A., MADICO, G.E., GLAD, S.M.,PAIGEN, B., QUINN, T.C., and EPSTEIN, S.E. (2001). Athero-sclerosis in apoE knockout mice infected with multiple pathogens.J. Infect. Dis. 183, 226–231.

51. GIRON-GONZALEZ, J.A., MORAL, F.J., ELVIRA, J., GARCIA-GIL, D., GUERRERO, F., GAVILAN, I., and ESCOBAR, L.(2000). Consistent production of a higher Th1:Th2 cytokine ratioby stimulated T cells in men compared with women. Eur. J. En-docrinol. 143, 31–36.

52. HANNA, N., HANNA, I., HLEB, M., WAGNER, E.,DOUGHERTY, J., BALKUNDI, D., PADBURY, J., andSHARMA, S. (2000). Gestational age-dependent expression of IL-10 and its receptor in human placental tissues and isolated cy-totrophoblasts. J. Immunol. 164, 5721–5728.

53. KRUSE, N., GRIEF, M., MORIABADI, N.F., MARX, L.,TOYKA, K.V., and RIECKMANN, P. (2000). Variations in cyto-kine mRNA expression during normal human pregnancy. Clin.Exp. Immunol. 119, 317–322.

54. RAGHUPATHY, R. (2001). Pregnancy: success and failure withinthe Th1/Th2/Th3 paradigm. Semin. Immunol. 13, 219–227.

55. WILDER, R.L. (1998). Hormones, pregnancy, and autoimmunediseases. Ann. NY Acad. Sci. 840, 45–50.

56. SAITO, S., FOEGH, M.L., MOTOMURA, N., LOU, H., KENT,K., and RAMWELL, P.W. (1998). Estradiol inhibits allograft-in-ducible major histocompatibility complex class II antigen expres-sion and transplant arteriosclerosis in the absence of immunosup-pression. Transplantation 66, 1424–1431.

Address reprint requests to:Dr. Alan Daugherty

University of KentuckySanders Brown, Room 424Lexington, KY 40536-0230

Tel: (859) 323-3996, ext. 290Fax: (859) 257-9166

E-mail: adaugh@uky.edu

Received 30 January 2002/Accepted 1 March 2002

WHITMAN ET AL.670

This article has been cited by:

1. Iryna Voloshyna, Michael J. Littlefield, Allison B. Reiss. 2013. Atherosclerosis and interferon-γ: Newinsights and therapeutic targets. Trends in Cardiovascular Medicine . [CrossRef]

2. Tian Ma, Qi Gao, Faliang Zhu, Chun Guo, Qun Wang, Fei Gao, Lining Zhang. 2013. Th17 cells andIL-17 are involved in the disruption of vulnerable plaques triggered by short-term combination stimulationin apolipoprotein E-knockout mice. Cellular and Molecular Immunology 10:4, 338-348. [CrossRef]

3. Bart Legein, Lieve Temmerman, Erik A. L. Biessen, Esther Lutgens. 2013. Inflammation and immunesystem interactions in atherosclerosis. Cellular and Molecular Life Sciences . [CrossRef]

4. R. E. J. Mitchel, M. Hasu, M. Bugden, H. Wyatt, G. Hildebrandt, Y-X. Chen, N. D. Priest, S. C. Whitman.2013. Low-Dose Radiation Exposure and Protection Against Atherosclerosis in ApoE –/– Mice: TheInfluence of P53 Heterozygosity. Radiation Research 179:2, 190-199. [CrossRef]

5. Giovanni Almanzar, Robert Öllinger, Julianna Leuenberger, Elisabeth Onestingel, Barbara Rantner, SarahZehm, Benno Cardini, Ruurd van der Zee, Cecilia Grundtman, Georg Wick. 2012. Autoreactive HSP60epitope-specific T-cells in early human atherosclerotic lesions. Journal of Autoimmunity 39:4, 441-450.[CrossRef]

6. E. Maganto-Garcia, M. Tarrio, A.H. LichtmanMouse Models of Atherosclerosis . [CrossRef]7. M. Benagiano, F. Munari, A. Ciervo, A. Amedei, S. R. Paccani, F. Mancini, M. Ferrari, C. Della Bella,

C. Ulivi, S. D'Elios, C. T. Baldari, D. Prisco, M. de Bernard, M. M. D'Elios. 2012. Chlamydophilapneumoniae phospholipase D (CpPLD) drives Th17 inflammation in human atherosclerosis. Proceedingsof the National Academy of Sciences . [CrossRef]

8. Jessica Gage, Mirela Hasu, Mohamed Thabet, Stewart C. Whitman. 2012. Caspase-1 Deficiency DecreasesAtherosclerosis in Apolipoprotein E-Null Mice. Canadian Journal of Cardiology . [CrossRef]

9. Michael V. Autieri. 2012. Pro- and Anti-Inflammatory Cytokine Networks in Atherosclerosis. ISRNVascular Medicine 2012, 1-17. [CrossRef]

10. Mirela Hasu, Mohamed Thabet, Nancy Tam, Stewart C. Whitman. 2011. Specific loss of Toll-like receptor2 on bone marrow derived cells decreases atherosclerosis in LDL receptor null mice* The senior author,Stewart C. Whitman, passed away on 19 February 2010. The manuscript has been communicated byRoss W. Milne (e-mail: rmilne@ottawaheart.ca) and Yves L. Marcel (e-mail: ylmarcel@ottawaheart.ca),University of Ottawa Heart Institute, 40 Ruskin Street, Ottawa, ON K1Y 4W7, Canada. Canadian Journalof Physiology and Pharmacology 89:10, 737-742. [CrossRef]

11. Zhao Zhao, Yue Wu, Manli Cheng, Yuqiang Ji, Xiaobo Yang, Ping Liu, Shaobin Jia, Zuyi Yuan. 2011.Activation of Th17/Th1 and Th1, but not Th17, is associated with the acute cardiac event in patientswith acute coronary syndrome. Atherosclerosis 217:2, 518-524. [CrossRef]

12. R. E. J. Mitchel, M. Hasu, M. Bugden, H. Wyatt, M. P. Little, A. Gola, G. Hildebrandt, N. D. Priest,S. C. Whitman. 2011. Low-Dose Radiation Exposure and Atherosclerosis in ApoE −/− Mice. RadiationResearch 175:5, 665-676. [CrossRef]

13. Maureen McMahon, Bevra H Hahn, Brian J Skaggs. 2011. Systemic lupus erythematosus andcardiovascular disease: prediction and potential for therapeutic intervention. Expert Review of ClinicalImmunology 7:2, 227-241. [CrossRef]

14. Göran K Hansson, Andreas Hermansson. 2011. The immune system in atherosclerosis. NatureImmunology 12:3, 204-212. [CrossRef]

15. Yanice V. Mendez-Fernandez, Bonnie G. Stevenson, Cody J. Diehl, Nicole A. Braun, Nekeithia S. Wade,Roman Covarrubias, Sander van Leuven, Joseph L. Witztum, Amy S. Major. 2011. The inhibitory FcγRIIbmodulates the inflammatory response and influences atherosclerosis in male apoE−/− mice. Atherosclerosis214:1, 73-80. [CrossRef]

16. Rutger J. Wierda, Sacha B. Geutskens, J. Wouter Jukema, Paul H.A. Quax, Peter J. Van Den Elsen.2010. Epigenetics in atherosclerosis and inflammation. Journal of Cellular and Molecular Medicine 14:6a,1225-1240. [CrossRef]

17. Min Guo, Xiaobo Mao, Qingwei Ji, Mingjian Lang, Songnan Li, Yudong Peng, Wei Zhou, Bo Xiong,Qiutang Zeng. 2010. Inhibition of IFN Regulatory Factor-1 Down-Regulate Th1 Cell Function in Patientswith Acute Coronary Syndrome. Journal of Clinical Immunology 30:2, 241-252. [CrossRef]

18. J. A. Kelly, M. E. Griffin, R. A. Fava, S. G. Wood, K. A. Bessette, E. R. Miller, S. A. Huber, C. J. Binder,J. L. Witztum, P. M. Morganelli. 2010. Inhibition of arterial lesion progression in CD16-deficient mice:evidence for altered immunity and the role of IL-10. Cardiovascular Research 85:1, 224-231. [CrossRef]

19. John Andersson, Peter Libby, Göran K. Hansson. 2010. Adaptive immunity and atherosclerosis. ClinicalImmunology 134:1, 33-46. [CrossRef]

20. Xian-Cheng Jiang, Calvin Yeang, Zhiqiang Li, Mahua Chakraborty, Jing Liu, Hongqi Zhang, Yifan Fan.2009. Sphingomyelin biosynthesis: its impact on lipid metabolism and atherosclerosis. Clinical Lipidology4:5, 595-609. [CrossRef]

21. Ning Xiao, Miao Yin, Liang Zhang, Xuebin Qu, Hui Du, Xiaoming Sun, Liufeng Mao, Guocheng Ren,Cong Zhang, Yue Geng. 2009. Tumor necrosis factor-alpha deficiency retards early fatty-streak lesion byinfluencing the expression of inflammatory factors in apoE-null mice. Molecular Genetics and Metabolism96:4, 239-244. [CrossRef]

22. Ahmed Soliman, Patrick Kee. 2008. Experimental models investigating the inflammatory basis ofatherosclerosis. Current Atherosclerosis Reports 10:3, 260-271. [CrossRef]

23. Stephanie Schulte, Galina K. Sukhova, Peter Libby. 2008. Genetically Programmed Biases in Th1 andTh2 Immune Responses Modulate Atherogenesis. The American Journal of Pathology 172:6, 1500-1508.[CrossRef]

24. R. Kleemann, S. Zadelaar, T. Kooistra. 2008. Cytokines and atherosclerosis: a comprehensive review ofstudies in mice. Cardiovascular Research 79:3, 360-376. [CrossRef]

25. L. Rogers, S. Burchat, J. Gage, M. Hasu, M. Thabet, L. Wilcox, T. A. Ramsamy, S. C. Whitman. 2008.Deficiency of invariant V 14 natural killer T cells decreases atherosclerosis in LDL receptor null mice.Cardiovascular Research 78:1, 167-174. [CrossRef]

26. Maureen McMahon, Bevra H Hahn. 2007. Atherosclerosis and systemic lupus erythematosus —mechanistic basis of the association. Current Opinion in Immunology 19:6, 633-639. [CrossRef]

27. Victoria L. King, Lisa A. Cassis, Alan Daugherty. 2007. Interleukin-4 Does Not Influence Development ofHypercholesterolemia or Angiotensin II-Induced Atherosclerotic Lesions in Mice. The American Journalof Pathology 171:6, 2040-2047. [CrossRef]

28. Yinong Wang, Usman Ahmad, Tai Yi, Liping Zhao, Marc I. Lorber, Jordan S. Pober, George Tellides.2007. Alloimmune-Mediated Vascular Remodeling of Human Coronary Artery Grafts in ImmunodeficientMouse Recipients Is Independent of Preexisting Atherosclerosis. Transplantation 83:11, 1501-1505.[CrossRef]

29. Elisabetta Profumo, Claudia Esposito, Brigitta Buttari, Maria Elena Tosti, Elena Ortona, Paola Margutti,Alessandra Siracusano, Angela Sposato, Alessandro Costanzo, Raffaele Capoano, Bruno Salvati, RacheleRiganò. 2007. Intracellular expression of cytokines in peripheral blood from patients with atherosclerosisbefore and after carotid endarterectomy. Atherosclerosis 191:2, 340-347. [CrossRef]

30. Michael E. Rosenfeld, Alan D. AttieGene Therapy and Cardiovascular Diseases . [CrossRef]31. Koen Vandenbroeck, An GorisThe IFNG–IL26–IL22 Cytokine Gene Cluster 157-174. [CrossRef]32. Amy S. Major, Sebastian Joyce, Luc Van Kaer. 2006. Lipid metabolism, atherogenesis and CD1-restricted

antigen presentation. Trends in Molecular Medicine 12:6, 270-278. [CrossRef]33. Göran K. Hansson, Anna-Karin L. Robertson, Cecilia Söderberg-Nauclér. 2006. INFLAMMATION

AND ATHEROSCLEROSIS. Annual Review of Pathology: Mechanisms of Disease 1:1, 297-329. [CrossRef]

34. Stewart C. Whitman, Tanya A. Ramsamy. 2006. Participatory role of natural killer and natural killer Tcells in atherosclerosis: lessons learned from in vivo mouse studiesThis paper is one of a selection of paperspublished in this Special Issue, entitled Young Investigator's Forum. Canadian Journal of Physiology andPharmacology 84:1, 67-75. [CrossRef]

35. K RAKESH, D AGRAWAL. 2005. Cytokines and growth factors involved in apoptosis and proliferationof vascular smooth muscle cells. International Immunopharmacology 5:10, 1487-1506. [CrossRef]

36. Göran K. Hansson. 2005. Inflammation, Atherosclerosis, and Coronary Artery Disease. New EnglandJournal of Medicine 352:16, 1685-1695. [CrossRef]

37. Pål Aukrust, Arne Yndestad, Torgun Wæhre, Lars Gullestad, Bente Halvorsen, Jan Kristian Damås. 2005.Inflammation in coronary artery disease: potential role for immunomodulatory therapy. Expert Review ofCardiovascular Therapy 3:6, 1111. [CrossRef]

38. Offer Galili, Joerg Herrmann, Julie Woodrum, Katherine J. Sattler, Lilach O. Lerman, Amir Lerman.2004. Adventitial vasa vasorum heterogeneity among different vascular beds. Journal of Vascular Surgery40:3, 529-535. [CrossRef]

39. Christoph J. Binder, Karsten Hartvigsen, Mi-Kyung Chang, Marina Miller, David Broide, Wulf Palinski,Linda K. Curtiss, Maripat Corr, Joseph L. Witztum. 2004. IL-5 links adaptive and natural immunityspecific for epitopes of oxidized LDL and protects from atherosclerosis. Journal of Clinical Investigation114:3, 427-437. [CrossRef]

40. A.-K. L. Robertson, X. Zhou, B. Strandvik, G. K. Hansson. 2004. Severe Hypercholesterolaemia Leadsto Strong Th2 Responses to an Exogenous Antigen. Scandinavian Journal of Immunology 59:3, 285-293.[CrossRef]

41. Tamikazu Niwa, Hisayasu Wada, Hazuki Ohashi, Naoki Iwamoto, Hirotoshi Ohta, Hirokazu Kirii,Hidehiko Fujii, Kuniaki Saito, Mitsuru Seishima. 2004. Interferon-.GAMMA. Produced by BoneMarrow-derived Cells Attenuates Atherosclerotic Lesion Formation in LDLR-deficient Mice. Journal ofAtherosclerosis and Thrombosis 11:2, 79-87. [CrossRef]

42. Anna-Karin L. Robertson, Mats Rudling, Xinghua Zhou, Leonid Gorelik, Richard A. Flavell, Göran K.Hansson. 2003. Disruption of TGF-β signaling in T cells accelerates atherosclerosis. Journal of ClinicalInvestigation 112:9, 1342-1350. [CrossRef]

43. Piers Davenport, Peter G. Tipping. 2003. The Role of Interleukin-4 and Interleukin-12 in the Progressionof Atherosclerosis in Apolipoprotein E-Deficient Mice. The American Journal of Pathology 163:3,1117-1125. [CrossRef]

44. Koen Vandenbroeck, An Goris. 2003. Cytokine gene polymorphisms in multifactorial diseases: gatewaysto novel targets for immunotherapy?. Trends in Pharmacological Sciences 24:6, 284-289. [CrossRef]