Elsevier

Atherosclerosis

Volume 153, Issue 2, December 2000, Pages 403-411
Atherosclerosis

Interleukin (IL)-4 deficiency does not influence fatty streak formation in C57BL/6 mice

https://doi.org/10.1016/S0021-9150(00)00418-4Get rights and content

Abstract

Abundant data is present to implicate oxidatively modified low-density lipoprotein (oxLDL) in enhanced atherogenesis. Among the factors involved in LDL oxidation, an important role has been attributed to human 15-lipoxygenase (LO) and its murine analog 12-LO. The expression of these peroxidizing enzymes is under the control of cytokines, the principal of which is IL-4. In the present study we tested the hypothesis that knocking out the IL-4 gene from C57BL/6 mice would result in suppression of fatty streaks. For this purpose, we have fed 45 female IL-4 transgenic knockout (IL-4T KO) and 45 wild-type (WT) mice an atherogenic diet for 15 weeks. Consecutive determinations of the lipid profile from both study groups were performed at monthly intervals, and fatty streak formation was assessed at the aortic sinus level, upon sacrifice. The two study groups did not differ significantly with respect to the lipid profile or the uptake and degradation of iodinated oxLDL by their peritoneal macrophages. We found that the endogenous deficiency of IL-4 did not confer protection from early atherosclerosis in the IL-4T KO as compared to their WT littermates (determined at the aortic sinus). Immunohistochemical studies, Western blots and 12/15-LO activity assays revealed the presence and activity of 12/15-LO in macrophages of WT mice as well as in IL-4T KO mice. Both did not differ significantly between the study groups. The data from this study imply that deficiency in IL-4 does not affect early atherosclerosis in C57BL/6 mice fed a high-cholesterol diet.

Introduction

In recent years, evidence has been presented to support the role of oxidative modification of low-density lipoprotein (oxLDL) in promoting atherogenesis [1], [2]. The direct visualization of oxLDL within animal and human atherosclerotic plaques has been demonstrated by immunohistochemical methods [reviewed in Ref. [2]]. Additionally, several antioxidants have been shown to suppress the progression of atherosclerosis in Watanabe heritable hyperlipidemic rabbits (WHHL) [3]. Indirect support for the atherogenicity of oxLDL stems from in vitro studies demonstrating its rapid entry and processing by macrophages, its activation of endothelial cells and its chemotactic properties — all considered contributory to the atherosclerosis process [reviewed in [2], [4]].

The processes involved in LDL oxidation in vivo and their relative importance have still not been elucidated, to date. Cellular elements such as smooth muscle cells, macrophages and endothelial cells (EC), which form the atherosclerotic plaque, can promote LDL oxidation. Yet, it has been suggested that smooth muscle cells act predominantly by forming superoxide anion [5], whereas cellular lipoxygenases appear to contribute to oxidation by macrophages and EC [6], [7].

Lipoxygenases are non-heme iron containing enzymes involved in lipid peroxidation by hydroperoxide formation from fatty acid substrates. The 15-LO is discerned by its ability to oxygenate polyenoic fatty acids esterified to various membrane lipids [8] and lipoproteins [9]. The human 15-LO was cloned by Sigal et al. [10]. It was subsequently reported that the 15-LO mRNA localizes in rabbit and human atherosclerotic plaque [11], [12]. These observations imply that this peroxidizing enzyme may play a principal role in enhancing atherosclerosis. Recently, we have provided direct proof for the proatherogenicity of 15-LO by creating a double transgenic mouse deficient in LDL-receptor and overexpressing human 15-LO in the vessel walls [13]. These mice developed enhanced atherosclerosis and their LDL was more susceptible to ex vivo oxidation when compared with their LDL-receptor deficient littermates. The leukocyte 12/15-LO shares high degree of structural homology with the 15-LO and is found in the mouse [14], pig [15] and cattle [16]. The current data on the involvement of leukocyte 12/15 and 15 LO in atherogenesis has recently been summarized [17].

IL-4, a cytokine with a key role in the commitment of CD4 T-cells to the Th2 lineage is the product of activated T lymphocytes, mast cells, basophils and some populations of natural killer cells [18]. IL-4 is an anti-inflammatory cytokine due to several actions: inhibition of IFN-gamma production, suppression of IFN-gamma mediated macrophage activation, reduction of procoagulant activity expression by activated endothelial cells and amelioration of tissue damage induced by neutrophil influx [reviewed in [18]]. The regulation of expression of 15-LO in human monocytes was shown to be influenced by interleukin-4 (IL-4) [19]. Similarly, it has recently demonstrated that IL-4 (as well as IL-13) induces upregulation of murine macrophage 12/15 lipoxygenase activity [20] implicating involvement of the transcription factor STAT6 [21].

In the current study we wished to examine the effect of knocking out the IL-4 gene in C57BL/6 mice on atherosclerosis development. We postulated that the lack of IL-4 might alter several pathways in early atherogenesis (i.e. reduction in 12/15-LO expression) and concomitantly result in suppression of atherosclerosis. However, we have found that similar atherosclerotic lesion size was evident in both IL-4T-knockout and the wild-type controls, in parallel with the similar 12/15-LO protein content and activity obtained from mice of the corresponding study groups.

Section snippets

Animals

Forty-five transgenic IL-4 gene-knockout (IL-4T KO) C57BL/6J and 45 control C57BL/6J mice (obtained from the Charles River Labs). All mice were females at ages of 3–5 weeks.

Diet

The ‘Paigen’ atherogenic diet containing ∼17% total fat and 1.25% cholesterol (Teklad Premier Laboratory Diet No. TD 90221) was used throughout the study.

Experimental design

All mice were fed ad libitum with the atherogenic diet, starting from the age of 3–5 weeks, for 15 weeks. The mice were bled from their retrorbital plexus at baseline, 4,

Results

All mice appeared healthy throughout the experiment and weight gain was similar in both study groups.

Discussion

In the current study we wished to test the hypothesis that knocking out the IL-4 gene in C57BL/6 mice would result in inhibition of early atherosclerosis. We reasoned that since IL-4 is an important inducer of 12/15-LO expression [19], [20], [21], a deficiency of this cytokine might significantly decrease the level of the murine enzyme and consequently reduce the production of the atherogenic oxLDL.

Our results show that both IL-4T KO and WT C57BL/6 mice developed a similar extent of early

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