Interleukin (IL)-4 deficiency does not influence fatty streak formation in C57BL/6 mice
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
References (39)
The oxidation hypothesis of atherosclerosis
Lancet
(1994)- et al.
The role of sulfur containing amino acids in superoxide production and modification of low density lipoprotein by arterial smooth muscle cells
J. Biol. Chem.
(1987) - et al.
Evidence for a dominant role of lipoxygenase(s) in oxidation of LDL by mouse peritoneal macrophages
J. Lipid Res.
(1991) - et al.
Occurrence of lipoxygenase products in membranes of rabbit reticulocytes lipoxygenase in the maturation of red cells
J. Biol. Chem.
(1990) - et al.
Enzymatic modification of low density lipoprotein by purified lipoxygenase plus phospholipase A2 mimics cell mediated oxidative modification
J. Lipid Res.
(1988) - et al.
cDNA cloning, expression, mutagenesis of c-terminal isoleucine, genomic structure, and chromosomal localization of murine 12-lipoxygenases
J. Biol. Chem.
(1994) - et al.
Identification of a novel arachidonate 12-lipoxygenase in bovine tracheal epithelial cells distinct from leukocyte and platelet form of the enzyme
J. Biol. Chem.
(1990) - et al.
Lymphocyte responses and cytokines
Cell
(1994) - et al.
Interleukin-4 and 13 induce upregulation of the murine macrophage 12/15-lipoxygenase activity: Evidence for the involvement of transcription factor STAT6
Blood
(1998) - et al.
Receptor mediated endocytosis of low density lipoprotein in cultured cells
Meth. Enzymol.
(1983)
Beyond cholesterol: modification of low density lipoprotein that increases its atherogenicity
N. Engl. J. Med.
The effect of probucol on the progression of atherosclerosis in mature Watanabe heritable hypercholesterolemic rabbits
Br. J. Pharmacol.
The pathogenesis of atherosclerosis: a perspective for the 1990s
Nature
A role for endothelial cell lipoxygenase in the oxidative modification of low density lipoprotein
Proc. Natl. Acad. Sci. USA
Molecular cloning and primary structure of human 15-lipoxygenase
Biochem. Biophys. Res. Commun.
Colocalization of 15-lipoxygenase mRNA and protein with epitopes of oxidized low density lipoprotein in macrophage rich areas of atherosclerotic lesions
Proc. Natl. Acad. Sci. USA
Induction of 15-LO mRNA and protein in early atherosclerotic lesions
Circulation
Cloning and sequence analysis of the cDNA of arachidonate 12-lipoxygenase of porcine leukocytes
Proc. Natl. Acad. Sci. USA
Cited by (30)
Inflammatory biomarkers for predicting cardiovascular disease
2013, Clinical BiochemistryAssociation of IL-4 receptor gene polymorphisms with high density lipoprotein cholesterol
2012, CytokineCitation Excerpt :Several studies have documented that genetic polymorphisms at loci encoding IL-4 and IL-4R can influence the activity of these genes or their products, and are associated with genetic predisposition to diseases [19,32–34]. Georgea et al. studied the influence of IL-4 to fatty streak formation using IL-4 knockout mice and found that HDL and triglycerides in the IL-4-deficient mice were higher [35], which further provides evidence that IL-4 is involved in lipid metabolism. In support of the above findings, our recent study also revealed a significant association between IL-4 genotypes and HDL-C levels [36].
Inflammatory markers and restenosis in peripheral percutaneous angioplasty with intravascular stenting: Current concepts
2011, Annals of Vascular SurgeryCitation Excerpt :Information regarding other anti-inflammatory cytokines is sparse and somewhat conflicting. Deficiency of IL-4 did not reduce or increase protection from early atherosclerosis in the IL-4 null mouse.80 Although several studies in cultured cells demonstrated convincingly that IL-11 has anti-inflammatory effects on leukocyte function, including inhibition of TNF-α, IL-1β, and interferon-γ synthesis, no in vivo studies have been reported regarding the ability of this cytokine to influence the vascular response to atherosclerosis or mechanical injury.
Association of interleukin-4 promoter polymorphisms in Taiwanese patients with type 2 diabetes mellitus
2010, Metabolism: Clinical and ExperimentalCitation Excerpt :Elbe-Bürger et al [40] further demonstrated that the adipocyte layer in the dermis is reduced in IL-4 transgenic mice. Moreover, by exploring the influence of IL-4 to fatty streak formation, George et al [41] found that HDL and triglycerides in IL-4–deficient mice were higher. The above studies indicate that local microenvironmental expression of IL-4 is involved in lipid metabolism and eventually the atherogenic process.
Interleukin-4 does not influence development of hypercholesterolemia or angiotensin II-induced atherosclerotic lesions in mice
2007, American Journal of PathologyCitation Excerpt :A small number of studies have investigated the effects of IL-4 deficiency on lesion size. These include studies in wild-type C57BL/6 mice in which atherosclerotic lesion formation was promoted by feeding a diet enriched in saturated fat, cholesterol, and cholate or through the inflammatory stimuli of incomplete Freund's adjuvant, heat shock protein-65, and Mycobacterium tuberculosis.36,37 IL-4 deficiency had no effect on the lesions formed during feeding of the cholate-containing diet or administration of incomplete Freund's adjuvant.
Serum levels of interleukin-18 in patients with stable and unstable angina pectoris
2005, International Journal of Cardiology