The role of interleukin-4 and interleukin-12 in the progression of atherosclerosis in apolipoprotein E-deficient mice.

American Journal of Pathology, Vol. 163, No. 3, September 2003 Copyright © American Society for Investigative Pathology The Role of Interleukin-4 and Interleukin-12 in the Progression of Atherosclerosis in Apolipoprotein E-Deficient Mice Piers Davenport and Peter G. Tipping From the Centre for Inflammatory Diseases, Department of Medicine, Monash University, Clayton, Victoria, Australia Accumulation of T cells and macrophages in atherosclerotic plaques and the formation of antibodies directed against plaque proteins suggests that adaptive immunity contributes to the development of atherosclerosis. The contribution of Th1 and Th2 helper cell subsets to atherogenesis was studied in a murine model by interbreeding apolipoprotein E-deficient (apoEⴚ/ⴚ) mice with mice deficient in key cytokines that drive either Th1 responses [interleukin (IL)-12] or Th2 responses (IL-4). Compared to apoEⴚ/ⴚ mice, apoEⴚ/ⴚ/IL-12ⴚ/ⴚ mice had a 52% reduction in plaque area in the aortic root at 30 weeks of age (P < 0.001). ApoEⴚ/ⴚ/IL-4ⴚ/ⴚ mice had a 27% reduction in plaque area compared to apoEⴚ/ⴚ mice (P < 0.05) at 30 weeks of age, but their plaques were significantly larger than in apoEⴚ/ⴚ/IL-12ⴚ/ⴚ mice at this stage (P < 0.05). By 45 weeks of age, there were no significant differences in lesion sizes in the aortic root between the strains, however apoEⴚ/ⴚ/IL-4ⴚ/ⴚ mice showed a 58% and 64% decrease in disease in their aortic arch compared to apoEⴚ/ⴚ (P < 0.05) and apoEⴚ/ⴚ/IL-12ⴚ/ⴚ (P < 0.05) mice, respectively, and a 78% decrease in thoracic lesions compared to apoEⴚ/ⴚ/ IL-12ⴚ/ⴚ (P < 0.05). This suggests that both Th1 and Th2 cytokines play roles throughout the development of atherosclerosis in various vascular sites in apoEⴚ/ⴚ mice. (Am J Pathol 2003, 163:1117–1125) Atherosclerosis has many of the hallmarks of a chronic immune inflammatory disease, although its pathogenesis is not fully understood. The role of resident smooth muscle cells, endothelial cells, and infiltrating cells such as macrophages in the development of atherosclerotic lesions has been extensively studied, but less so the role of infiltrating T lymphocytes and adaptive immunity. CD4⫹ and CD8⫹ T cells are present throughout the development of lesions in humans.1–3 The co-localization of T cells and macrophages within lesions,4 the expression of MHC class II molecules4,5 and interleukin (IL)-2 receptor4 and presence of CD40/CD40 ligand5 is consistent with the involvement of adaptive cell-mediated im- munity in atherogenesis. The observation that a significant proportion of T cells express activation markers and have heterogeneous T-cell receptor gene rearrangement patterns,6 suggests that T cells may be stimulated by a variety of local antigens such as oxidized low density lipoprotein (oxLDL),7,8 heat shock proteins,9,10 Chlamydia pneumoniae,11 or other microbes12 or are recruited by mechanisms independent of their antigen specificity. The contribution of adaptive immunity to the progression of atherosclerosis has been investigated using apolipoprotein E-deficient (apoE⫺/⫺) mice. These hypercholesterolemic mice spontaneously develop atherosclerosis with similar pathology to human disease.13,14 Interbreeding of these mice with immunodeficient recombination activating gene (RAG)-deficient mice or severe combined immunodeficient mice (SCID) has helped elucidate the role of adaptive immunity in the pathogenesis of atherosclerosis. When RAG-2⫺/⫺ mice, which lack functional T and B cells, are combined with apoE⫺/⫺ mice and fed a standard chow diet, a significant decrease in lesion size is observed.15 Similarly, RAG-1-deficient apoE⫺/⫺ mice showed a twofold decrease in aortic lesion size16 and apoE⫺/⫺/SCID/SCID mice showed a 73% decrease in fatty streak development.17 Transfer of CD4⫹ T cells from apoE⫺/⫺ mice to apoE⫺/⫺/SCID/SCID mice was associated with infiltration of these T cells into developing lesions and increased lesion size, suggesting acquisition of adaptive immunity accelerates atherosclerosis.17 Feeding immunodeficient mice with a moderate (Western diet) or a high-fat diet results in serum cholesterol levels many fold higher than in human disease and obscures the effects of adaptive immunity on the development of atherosclerosis.16,18 These effects may be partially explained by the ability of high cholesterol diets to modulate the Th1/Th2 immune response in apoE⫺/⫺ mice.19 Immunosuppression with cyclosporin A in C57BL/6 mice fed a high cholesterol diet caused an increase in early plaque development, although cyclosSupported by the National Health and Medical Research Council of Australia. P. G. T. is a Principal Research Fellow of National Health and Medical Research Council of Australia. Accepted for publication May 12, 2003. Address reprint requests to Piers Davenport, Monash University Department of Medicine, Level 5 Block E, Monash Medical Centre, 246 Clayton Rd., Clayton, Victoria, Australia 3168. E-mail: . 1117 1118 Davenport and Tipping AJP September 2003, Vol. 163, No. 3 porin-induced endothelial cell injury may contribute to this response.20 Plaque T cells are primarily of the T-helper 1 (Th1) subtype, secreting cytokines such as interferon (IFN)-␥, IL-2, and tumor necrosis factor-␣ and -␤,21–23 which are involved in macrophage activation and inflammation.12 IL-12, produced by many cell types including plaque macrophages, is important for Th1 differentiation21 and stimulates proliferation and differentiation of natural killer and T cells.24 IL-12 has also been shown to affect humoral responses by switching the immunoglobulin isotype to IgG2a in mice.25 IL-12 is expressed in the aortas of young apoE⫺/⫺ mice and administration of IL-12 increases plasma levels of IgG2a and IgM anti-oxLDL antibodies in the blood and accelerates development of atherosclerotic lesions in apoE⫺/⫺ mice.26 Th2 type cytokines, including IL-4, IL-5, and IL-10 are also expressed in human plaques but are somewhat less abundant than Th1 type cytokines.21,22 IL-4 is the major cytokine directing Th2 differentiation of Th0 cells.27 It is expressed by T cells in atheroma of severely hypercholesterolemic apoE⫺/⫺ mice,20 although T-cell clones generated from human atheromatous plaques seldom express a Th2 profile of cytokines (high IL-4 and low IFN-␥).28 IL-4 is produced by macrophages, mast cells, and others29,30 and promotes synthesis of IgE31 and allergic responses. It has the ability to inhibit Th1 responses, reduce macrophage activation, and IFN-␥ production and reduce procoagulant activity expression by endothelial cells.29,30 IL-4 also upregulates expression of the oxLDL-binding scavenger receptor32 and inhibits inducible nitric oxide synthase and cyclooxygenase 2.33,34 The contribution of Th1 and Th2 helper cell responses to the development of atherosclerosis remains unclear. This question was addressed in the current study by examining the development of atherosclerosis in apoE⫺/⫺ mice deficient in key cytokines responsible for (...truncated)