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(Circulation. 2001;103:2610.)
© 2001 American Heart Association, Inc.

Basic Science Reports

T Helper–Cell Phenotype Regulates Atherosclerosis in Mice Under Conditions of Mild Hypercholesterolemia

S. A. Huber, PhD; P. Sakkinen, MD; C. David, MD; M. K. Newell, PhD; R. P. Tracy, PhD

From the Departments of Pathology (S.A.H., P.S., R.P.T.), Biochemistry (R.P.T.), and Medicine (M.K.N.), University of Vermont, Burlington, and the Department of Immunology, Mayo Clinic, Rochester, Minn (C.D.).

Correspondence to Sally Ann Huber, PhD, Department of Pathology, University of Vermont, 208 S Park Dr, Suite 2, Colchester, VT 05446. E-mail shuber{at}salus.uvm.edu

	Abstract

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Background—T cells are implicated in atherosclerosis, but little is known about the genetic control or molecular pathways, especially under conditions of mild hypercholesterolemia.

Methods and Results—BALB/c mice, making a CD4+ Th2 (IL-4+) cell response, express both MHC class II antigens (IA^d, IE^d) and are atherosclerosis-resistant. C57Bl/6 mice produce a CD4+ Th1 (interferon [IFN]+) response, express IA^b but no IE, and are atherosclerosis-prone. To evaluate T helper–cell phenotype in fatty streak formation, wild-type C57Bl/6 mice (IA^b+IE-) and transgenic mice, either AB^o, IA^b-IE-; ABE, IA-IE^k+; or Bl.Tg.E, IA^b+IE^k+, were fed a high-cholesterol diet for 16 weeks and evaluated histomorphometrically for aortic lesions. Lesion size in AB^o, ABE, and Bl.Tg.E strains was decreased by 54%, 79%, and 82%, respectively, compared with wild-type, correlating with decreased Th1 and increased Th2 expression and suggesting that T helper–cell phenotype is important in fatty lesion development. Decreasing Th1 cells by antibodies (-CD4) or cytokines (IL-4) also caused 80% reductions in lesion size. Immunohistology revealed IFN-, but not IL-4, colocalized with activated macrophages. Confirming these findings in a different mouse strain, BALB/c Stat 6 knockout mice (Th2 cell–deficient) developed aortic lesions comparable to C57Bl/6 mice on the same diet.

Conclusions—In mildly hypercholesterolemic C57Bl/6 mice, presence of IA^b and absence of IE regulated CD4+ T helper–cell phenotype; fatty lesions were proportional to IFN+ Th1 cells in both C57Bl/6 and BALB/c strains. IFN- may participate through macrophage activation, whereas IL-4 may act to limit Th1-cell response.

Key Words: arteriosclerosis • immunology • lipids

	Introduction

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Although some mouse strains are atherosclerosis-resistant (eg, BALB/c), susceptible mice such as C57Bl/6 develop aortic fatty lesions that are similar to the early coronary lesions that develop in humans (Stary grades I to III). Several mutations have been introduced into these mice that result in rapidly progressing lesions; eg, the commonly used apoE-knockout (apoE ko) mouse.¹ To explore whether inflammation was an integral part of fatty lesion development in the murine model system, we recently studied the effect of weekly interleukin-6 (IL-6) injections on C57Bl/6 and C57Bl/6 apoE ko mice and observed 2- to 5-fold increased lesion size.² The greater effect was observed in the wild-type C57Bl/6 strain, suggesting that the very high cholesterol levels observed in apoE ko mice (>1000 mg/dL) may at least partly mask an underlying inflammation-related mechanism.

IL-6 has important T-cell immunoregulatory effects,³ and T helper (Th) cells (CD4+) are found in both murine⁴ and human⁵ fatty lesions. This suggests that the IL-6–mediated effects we observed may have an immunological component. Although it has been reported that T cells do not play an important role in the development of murine fatty lesions under conditions of severe hypocholesterolemia,⁶ under certain conditions depletion of T cells significantly decreases lipid accumulation in the aorta,^{6 7 8} leaving open the hypothesis that T cells may be important in fatty lesion development under conditions of milder hypercholesterolemia.

CD4+ T-cell responses are, to a great degree, regulated by the class II major histocompatability complex (MHC) molecules on the surface of antigen-presenting cells, such as macrophages. In the mouse, there are 2 MHC class II molecules, IA and IE, each consisting of an and a ß chain. MHC class II alleles act as major genetic susceptibility elements in a variety of autoimmune conditions, such as insulin-dependent diabetes mellitus, rheumatoid arthritis, and systemic lupus erythematosus.^{9 10 11} Interestingly, the atherosclerosis-susceptible strain C57Bl/6, unlike the resistant strain BALB/c, expresses only IA antigen and has been reported to produce predominantly Th1 helper cells.¹² This leads us to hypothesize that atherosclerosis susceptibility in C57Bl/6 mice, under conditions of mild hypercholesterolemia, may be a result of immune deviation toward Th1-cell production.

Because C57Bl/6 and BALB/c mice may differ in many ways other than MHC expression, we used several MHC knockout and transgenic mice, all on the C57Bl/6 background, to address this question. We then extended this work to include BALB/c mice that had been genetically modified to alter their natural T helper–cell expression: Stat 4 knockout mice, which produce primarily a Th2 response, and Stat 6 knockout mice, which produce primarily a Th1 response.

	Methods

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Mice
BALB/c, BALB/c Stat 4 knockout (BALB/c Stat 4 ko), and BALB/c Stat 6 knockout (BALB/c Stat 6 ko) mice were purchased from Jackson Laboratories, Bar Harbor, Me. Genetically modified C57Bl/6 mice were bred and housed at the University of Vermont Animal Care Facility. C57Bl/6 CD4 knockout (CD4 ko) mice were obtained originally from Dr T. Mak, then from Jackson Laboratories. MHC class II transgenic mice have been described.^{13 14 15 16 17} Briefly, C57Bl/6 inherently lack MHC class II IE because of a naturally occurring defect in the E, gene making them class II IA+IE-. MHC class II knockouts (AB^o mice) were made by mutating the Aß loci by use of homologous recombination, thus disrupting the gene. Clones of the disrupted Aß gene were injected into C57Bl/6 blastocysts, and chimeric males were bred to C57Bl/6 females. Progeny were backcrossed to C57Bl/6.^{15 16} Animals expressing class II IE (IA+IE+ or IA-IE+) were made by injecting a cloned E^k gene from A/J mice into male pronuclei of F2 hybrids from C57Bl/6xSJL animals (Bl.Tg.E mice). After the 18th generation of backcrossing transgenic E^k mice to C57Bl/6, these animals were bred with MHC class II knockout mice (AB^o) to make the IA-IE+ strain (AB^oE mice). Thus, these animals were congenic to the C57Bl/6 parental strain except for variations in MHC class II gene expression.

Experimental Design
To study fatty lesion development in C57Bl/6 wild-type and modified mice, 3-week-old male mice were fed ad libitum either Teklad diet 7012 (5.67% fat, 0% cholesterol) or 96354 (20% fat, 1.5% cholesterol, 0.5% sodium cholate) for 15 weeks, in 3 separate experimental designs. In experiment 1, we used the following mice, on a high-fat diet: n=10 C57Bl/6 (IA+IE-), n=7 AB^o (IA-IE-), n=6 Bl.Tg.E (IA+IE+), and n=4 AB^oE. Ten age- and sex-matched control C57Bl/6 mice were maintained on the low-fat diet. In experiment 2, mice on high fat were n=10 C57Bl/6 and n=10 C57Bl/6 CD4 ko. In experiment 3, mice on high fat were n=9 C57Bl/6, n=6 C57Bl/6 treated with IL-4 (Pharmingen, 50 ng in PBS weekly), and n=4 C57Bl/6 treated with IL-12 (Pharmingen, 50 ng in PBS weekly).

In a separate experiment (experiment 4), the BALB/c (n=8), BALB/cStat 4 ko (n=5), and BALB/cStat 6 ko (n=8) mice were fed the high-fat diet for 15 weeks.

Plasma Lipids
Blood was collected by cardiac puncture into EDTA tubes. Plasma from individual mice was assayed for total cholesterol by anenzymatic method, and plasma from several animals in each group (n=2 to 5) was pooled for total and HDL cholesterol and triglyceride measurements. LDL cholesterol was calculated.¹⁸

Histology
The heart and ascending aorta including the aortic arch were removed and evaluated for atherosclerotic lesions according to the histomorphometric method of Plump et al¹⁹ with oil red O–stained serial sections, as we have previously described.² An average of the fatty lesion throughout the whole aortic sinus was obtained for each animal. Antibodies used for immunohistology were rat -Mac3, rat -IL-4, and rat -IFN-, all from Pharmingen.

Cell Surface and Intracellular Cytokine Staining
Antibodies for flow cytometry obtained from Pharmingen included PE- and FITC-rat IgG1 (clone R3-34); PerCP-rat IgG2a (clone R35-95); FITC-rat anti-mouse interferon- (IFN-; clone XMG 1.2); PE-rat anti-mouse IL-4 (clone BVD4-1D11); PerCP-rat anti-mouse CD4 (clone RM4-5); PE-mouse anti-IA^b (clone AF6-120.1); and PE-mouse anti-IE^k (clone 14-4-4S). Ascites antibodies to CD4 were made by standard methods.

Lymphoid cells were isolated by centrifugation of diluted blood (1:10 with RPMI 1640, Sigma Chemical Co) on Histopaque (Sigma) at 670g for 10 minutes. Spleens were removed, pressed through fine-mesh screens to produce single-cell suspensions, and washed in RPMI 1640 medium containing 5% FBS and antibiotics.

For cell surface staining, 1x10⁵ lymphocytes were incubated with 1:100 dilution of specific antibodies or isotype controls in PBS, 1% BSA (Sigma), 0.01% sodium azide for 30 minutes on ice. The cells were washed twice and fixed in 2% paraformaldehyde (EMS).

For intracellular staining of CD4⁺ cells, 1x10⁶ cells were cultured in medium containing 10 µg/mL brefelden A (Sigma) for 4 hours at 37°C with 5% CO₂. In some cases, peripheral cells were also stimulated by including PMA and ionomycin. Cells were washed and incubated with anti-CD4 for 30 minutes on ice. After fixation and washing, the cells were made permeable with PBS, 1% BSA, and 0.5% saponin for 10 minutes. The cells were incubated with FITC-rat anti–IFN-, PE-rat anti–IL-4, or PE/FITC-rat IgG1 isotype control for 30 minutes, washed, and resuspended. Staining was evaluated with a Coulter Epics Elite instrument with a single excitation wavelength of 488 nm and emission band filters for PerCP (670 nm), PE (575 nm), and FITC (525 nm). Data represent positive cells minus the isotype control values.

Statistics
Mean±SEM values are presented graphically. All statistical testing for differences between groups was done by nonparametric methodsbecause of small sample sizes²⁰ (Kruskal-Wallis test for overall differences and Mann-Whitney test for pairwise comparisons). Correlation was expressed as Pearson correlation coefficient. Statistical significance was set at a value of P0.05.

	Results

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Lipid Levels
On high-fat diets, no significant differences in total cholesterol or lipoprotein subfraction levels were observed between C57Bl/6 mice and any of the transgenic C57Bl/6 groups (Figure 1). The overall cholesterol value was 172 mg/dL, representing a mild hypercholesterolemia, compared with 78 mg/dL (C57Bl/6 wild-type; low fat). Total cholesterol levels were slightly higher in BALB/c mice, 196 mg/dL. Total cholesterol levels in BALB/c Stat 6 ko mice were again slightly higher than in BALB/c controls (253 mg/dL; P<0.05).

View larger version (31K): [in this window] [in a new window]   Figure 1. Plasma cholesterol levels in mice from experiments 1 and 4. Experiment 1: C57Bl/6 mice were fed high-fat diet (first 5 bars) or low-fat diet (last bar) for 15 weeks. Animals were bled by intracardiac puncture at time of euthanasia with 0.1 mmol/L EDTA as anticoagulant. Results represent mean±SEM of 4 to 10 mice per group. No significant differences were observed among the high-fat diet groups (first 5 bars). C57Bl/6 mice fed low-fat diet were significantly different from others, P0.001.

T-Cell Phenotype Expression
In Table 1, IA and IE indicate the total number of cells positive for each MHC marker. As expected, in both the spleen and peripheral blood, the C57Bl/6 strains lack IE reactivity, whereas the IE transgenic mice exhibit restored IE expression. The IA knockout mice exhibited markedly reduced IA expression, but IA expression was not completely removed.

View this table: [in this window] [in a new window]   Table 1. Phenotypic Characterization of Lymphoid Cells from C57Bl/6 and Genetically Modified Mice in Experiment 1

Total CD4+ reactive cells were approximately equivalent in the different strains in both the spleen and blood samples, with the exception of the AB^o mice. The Th1/Th2 ratio differed as expected on the basis of IA and IE expression. The mice kept on a low-fat diet lacked immune activation.

Wild-type BALB/c mice (IA^d+IE^d+) resembled Bl.Tg.E mice having equivalent CD4+ Th1 and Th2 cells, Table 2. As expected, BALB/c Stat 6 knockout animals have strongly positive CD4+ Th1-cell responses but minimal CD4+ Th2 cells, whereas BALB/c Stat 4 knockout mice have the opposite phenotype.

View this table: [in this window] [in a new window]   Table 2. Phenotype Characteristics of Lymphoid Cells From BALB/c and Genetically Modified Mice in Experiment 4

Figure 2 gives representative flow diagrams of the BALB/c, BALB/c Stat 4 ko, and BALB/c Stat 6 ko mice staining for CD4, IFN-, and IL-4. Numbers in the upper right corners indicate the percentage of cells in each quadrant and clearly show the bias of BALB/c Stat 4 ko mice to a Th2 (IL4+) phenotype and BALB/c Stat 6 ko mice to a Th1 (IFN+) phenotype.

View larger version (39K): [in this window] [in a new window]   Figure 2. Intracellular cytokine staining of BALB/c mice. Peripheral blood lymphocytes from individual mice were cultured with PMA, ionomycin, and brefeldin A for 4 hours, surface-stained for CD4, and then fixed with paraformaldehyde. Plasma membrane was permeabilized with saponin, and cells were intracellularly stained for IFN- and IL-4. Numbers in upper right corners indicate percentage of peripheral blood lymphocytes in each quadrant.

Fatty Lesion Development
Experiment 1 in Figure 3 demonstrates the role of MHC class II antigen expression on atherosclerosis in C57Bl/6 and BALB/c mice. None of the C57Bl/6 mice on the low-fat diet showed any lipid accumulation in the aorta (data not shown). In contrast, C57Bl/6 mice with mild hypercholesterolemia developed fatty lesions. The lesions were typical for the murine model of atherosclerosis, with subendothelial accumulations of lipid-laden foam cells and variable amounts of extracellular lipid (Figure 4). Statistical testing indicated an overall difference between the groups (P0.001), and pairwise testing indicated that the C57Bl/6 wild-type mice developed significantly more fatty lesions than each of the other groups transgenic for class II MHC antigen expression. There was a significant correlation between remaining CD4+ cells and lesion size (r=0.907; P0.005) in mice treated with anti-CD4 antibodies.

View larger version (33K): [in this window] [in a new window]   Figure 3. Fatty lesion size in mice from experiments 1, 2, and 4. In experiment 1 (left), C57Bl/6, MHC class II transgenic mice and C57Bl/6 mice treated biweekly with monoclonal anti-CD4 were fed the high-fat diet for 15 weeks. C57Bl/6 mice fed the low-fat diet did not develop lesions (data not shown). In experiment 2 (middle), C57Bl/6 mice and C57Bl/6 CD4 ko mice were fed high-fat diet. In experiment 4 (right), BALB/c, BALB/c Stat 6 ko (Th1+), and BALB/c Stat 4 ko (Th2+) were fed high-fat diet. Lesion size for each mouse is an average of four 10-µm sections of proximal aorta. Each section was 80 µm distal to previous section, covering a total of 320 µm of aorta. Results represent mean±SEM of 4 to 10 mice per group. Statistical testing was done nonparametrically, with *P0.05.

View larger version (139K): [in this window] [in a new window]   Figure 4. Fatty lesions in C57Bl/6 and CD4-deficient mice with mild hypercholesterolemia: (A) C57Bl/6, (B) C57Bl/6 treated with anti-CD4 monoclonal antibody, or (C) C57Bl/6 CD4 ko mice. Magnification x16. Thick arrow in A demonstrates fatty depositions in subendothelial spaces of aorta; thick arrow in B demonstrates small amount of residual subendothelial lesion; no subendothelial stain was detected in C. Thin arrows in A and C demonstrate fat deposits outside of aorta. D, High magnification (x160) of region in A marked by thick arrow demonstrating both intracellular and extracellular subendothelial fat deposition.

C57Bl/6 CD4 ko mice have few fatty lesions compared with wild-type C57Bl/6 (CD4+) mice (experiment 2). Finally, although neither BALB/c mice nor transgenic BALB/c Stat 46 ko (Th2+) mice had aortic lesions, the BALB/c Stat 6 ko mice develop prominent lesions that closely resemble those of C57Bl/6 mice (experiment 4; Figures 3 and 5).

View larger version (130K): [in this window] [in a new window]   Figure 5. Fatty lesions in BALB/c mice with mild hypercholesterolemia. A, BALB/c; B, BALB/c Stat 4 ko (Th2+); and C, BALB/c Stat 6 ko (Th1+) mice. Magnification x16; arrowhead in C indicates fatty deposits.

Finally, we modulated T helper–cell expression through cytokine administration, Table 3. IL-4, produced by Th2 cells and known to downregulate Th1 expression, caused a marked decrease in Th1-cell expression in C57Bl/6 mice and a 90% decrease in lesion size. In contrast, IL-12, known to increase Th2 expression, had a small effect on C57Bl/6 Th2 expression (due to the IE defect) and no significant effect on lesion size.

View this table: [in this window] [in a new window]   Table 3. Cytokine Modulation of Th Cell Response in Early Atherosclerosis in C57Bl/6 Mice

Immunohistology (Figure 6) revealed the presence of IFN- and the absence of IL-4, suggesting the presence of Th1 cells and lack of Th2 cells in the lesions. Positive staining for Mac3 indicates the presence of activated macrophages at these same sites.

View larger version (49K): [in this window] [in a new window]   Figure 6. Immunohistological analysis of fatty lesions in C57Bl/6 mice. Sections were serially cut and stained as described in Methods section. Serial 10-µm sections were stained for (A) oil red O, (B) rat isotype antibody, (C) rat anti-Mac3, (D) rat anti–IL-4, and (E) rat anti–IFN-. Arrows indicate areas of positive staining. Magnification x60.

	Discussion

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We have demonstrated that MHC class II antigen expression regulates susceptibility to early atherosclerosis in atherosclerosis-prone C57Bl/6 mice under conditions of mild hypercholesterolemia. Specifically, IA expression, leading to Th1-cell development, appears to be proatherogenic, whereas IE expression, leading to Th2-cell development, appears to downregulate this process. This may occur through the cytokines expressed by these T helper cells, with IFN- being proatherogenic and IL-4 being antiatherogenic. BALB/c Stat 6 ko mice confirm the importance of IFN- to lesion development in a different species. Stat 6 is a transcription factor important in IL-4 expression.²¹ Thus, mice lacking Stat 6 show dominant CD4+ Th1-cell responses but few Th2 cells. BALB/c mice, which normally have relatively strong Th2-cell responses, show no fat deposition in the aortic sinus, whereas the BALB/c Stat 6 ko animals show substantial fat accumulation. This proatherogenesis may be augmented by IFN-–mediated macrophage activation,²² with subsequent lipid accumulation. Although interactions between MHC alleles have been shown to significantly affect susceptibility to diseases such as type 1 diabetes,¹¹ we believe that this is the first report of such regulation in atherosclerosis. With regard to our recent IL-6 findings,² we propose that IL-6 acts to increase lesion size through its ability to potentiate CD4+ T-cell differentiation.²³ This seems likely, because in the present study, lesion size was directly and strongly related to residual CD4+ cell number.

Th1 cell–derived IFN- may have other effects directly linked to early atherosclerosis. IFN- upregulates IL-1 and TNF- production, cytokines that are pivotal protein mediators of inflammation²⁴ and upregulate adhesion molecule expression on vascular endothelial cells.^{25 26} Gupta et al,²⁷ using compound apoE-deficient, IFN- receptor–deficient mice, showed that IFN- can potentiate murine atherosclerosis through both local effects in the arterial wall and systemic effects on plasma lipoproteins. Conversely, Th2-derived IL-4 inhibits proinflammatory cytokine release.²⁵ Alternatively, IA expression may allow the presentation of proatherogenic antigens, whereas IE does not. In any event, oxidized LDL, as suggested by others, should be considered an important candidate as the antigen responsible for T-cell activation.²⁸

The degree of hypercholesterolemia appears to be critical in defining the pathophysiology of early lesions in mice. Dansky et al⁶ failed to find a major role for T or B cells in severe hypercholesterolemia induced in apoE ko mice, but CD4+ T cells are known to infiltrate fatty lesions in these animals.⁷ An inhibition of atherosclerosis in CD4+-ablated, mildly hyperlipidemic C57Bl/6 mice has been observed⁸ (which we confirm), and in mildly hypercholesterolemic mice, there is a predominant Th1 response in the spleen, which switches to a Th2 response on severe hypercholesterolemia.²⁹ mRNA for Th1 cytokines has been identified in lesions from mildly lipidemic mice, and we demonstrate direct IFN- staining.²⁹ Infiltrating Th1 cells may play an important role in the autoimmune response to oxidized LDL and lesion development under conditions of mild hypercholesterolemia, which may more closely mimic the human condition.²⁹ Our data strongly support this position and suggest that Th1 cells may activate macrophages via IFN- and modulate by the presence of Th2 cells or the downstream effects of Th2-cell products such as IL-4.

Our data are not in agreement with those of Fyfe et al,³⁰ who compared C57Bl/6 wild-type mice to a variety of C57BL/6J mice carrying mutations resulting in various immune deficiencies, including class II MHC deficiency, and found little difference in lesion size among these groups. These investigators did not assess T-cell differentiation and maturation over time. Class II knockout mice have disrupted Aß and E genes, and homologous pairing (A/Aß and E/Eß) to form the complete class II -chain/ß-chain dimers cannot occur. Chimeric complexes, however, can and do occur (eg, A/Eß),³¹ and class II knockout mice do develop CD4+ T-cell maturation over time. Indeed, in our CD4 ko mice, we found some CD4+ cells in the spleen. Because the animals described above were older than the animals reported here, it is possible that they may have had even greater numbers of CD4+ cells, possibly enough to mask any difference in lesion development.

We used cholic acid in our high-fat diets, as have virtually all other investigators using murine atherosclerosis models. Bile salts, in particular ursodeoxycholate, in high quantities are known to have effects on the immune system.³² Although it is unlikely that the levels achieved by ingesting the relatively small amounts used in chow are of major importance, we cannot rule this out completely as a possible source of error.

We present data from 2 different strains of mice that suggest that T helper–cell expression may be critical in the development of early fatty lesions. If generalizable, there are several implications to our work. A possible role for MHC genotypes in human atherosclerosis has been proposed by others,³³ with associations apparently most pronounced in young men and older women.³⁴ Our animal model data are consistent with this position and suggest that the mechanism may be through immune deviation. Also, men generally have better Th1 cell–mediated cellular immunity than women, whereas women have superior Th2 cell–mediated humoral immunity.³⁵ Our findings are consistent with the well-known sex-based differences in atherosclerosis progression between men and women.

	Acknowledgments

 
This study was supported by NIH awards RO1-HL-58583 (Dr Huber), RO1-HL-46696 (Dr Tracy), T32-07594 (Dr Sakkinen), RO1-CA-24473 (Dr David), and RO1-AI-33470 (Dr Newell) and American Heart Association award 9750081 (Dr Huber).

Received December 8, 2000; revision received January 12, 2001; accepted January 23, 2001.

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