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Objective: Atherosclerosis is associated with immune responses to oxidized low-density lipoprotein (oxLDL). The presence of activated macrophages and T cells in lesions suggests that cell-mediated immune reactions are taking place during the disease process. However, the role of specific immune responses has remained unclear. We have previously shown that transfer of CD4+ T cells from apolipoprotein E knockout mice (apoE(-/-)) into immunodeficient apoE(-/-) scid/scid mice accelerates disease., Methods and Results: To test whether this effect is dependent on specific disease-associated antigens, purified CD4+ T cells from oxLDL-immunized mice were transferred into apoE(-/-) scid/scid mice. CD4+ T cells from mice immunized with a nonrelevant antigen, keyhole limpet hemocyanin (KLH), and naïve CD4+ T cells were used as controls. After 12 weeks, all mice that received T cells had larger lesions than untouched apoE(-/-) scid/scid controls. However, mice receiving CD4+ T cells from oxLDL immunized mice had substantially accelerated lesion progression compared with those receiving naive or KLH-primed T cells. Circulating levels of interferon-gamma were increased in proportion to the acceleration of atherosclerosis., Conclusions: These data show that adoptive transfer of purified CD4+ T cells from oxLDL-immunized mice accelerates atherosclerosis. They support the notion that Th1 cellular immunity is proatherogenic and identify oxLDL as a culprit autoantigen.

Objective: The proatherogenic effect of IL-18 is shown to be dependent on IFN-gamma production. It is believed that activated T cells play a proatherogenic role through secretion of IFN-gamma. However, recent studies in vitro have shown that macrophages, NK cells, and even vascular smooth muscle cells may also secrete IFN-gamma after stimulation by cytokines like IL-18. We therefore investigated whether cells other than activated T cells can play a proatherogenic role via IFN-gamma secretion under the stimulation of IL-18 in vivo., Methods and Results: SCID/apoE knockout mice were injected intraperitoneally with either IL-18 or phosphate-buffered saline 3 times per week for 7 weeks. Our results show that administration of IL-18 leads to 3-fold larger lesions and 2-fold higher circulating IFN-gamma despite the absence of T cells. In addition, increased IFN-gamma, accompanied by elevation of the scavenger receptor/chemokine CXCL16, was observed in both lesions and spleens. Furthermore, our findings revealed that macrophages, NK cells, and vascular cells were the source of IFN-gamma under the stimulation of IL-18 in the absence of T cells in vivo., Conclusions: The current data suggest that the proatherogenic effect of IL-18 can occur in the absence of T cells and that IFN-gamma secreted by macrophages, NK cells, and vascular cells is sufficient for the disease progression.

The C57BL/6J mouse strain is widely used as a common genomic background for many gene-modified murine models. However, few data on its clinical biochemistry are available. Therefore, we conducted a study to provide new protocols for serum biochemical screening and developed the reference range for a set of 13 analytes that pertain to lipoprotein metabolism, electrolyte balance, and data reflecting function of the heart, liver, kidneys, and pancreas. Male and female mice were studied, and blood samples were obtained at six and 20 weeks of age. Of 13 parameters studied, 12 were affected by age and sex. Briefly, male mice had higher triglycerides, cholesterol, high-density lipoprotein cholesterol, glucose, and amylase values. With age, mice of both sexes developed higher triglycerides and glucose concentrations, as well as aspartate and alanine transaminase activities. A significant difference between mice and humans was noted for amylase activity, which is extremely high in this healthy mouse strain. Therefore, we suggest that caution should be taken when data are interpreted to indicate gastrointestinal disease in murine models. The reference values for murine clinical biochemical analytes obtained during the study reported here should be useful for characterizing the biochemical phenotype of experimental mice.

Objective: Atherosclerosis is an inflammatory disease. Several chemokines are important for monocyte/macrophage and T-cell recruitment to the lesion. CXCL16 is a recently discovered chemokine that is expressed in soluble and transmembrane forms, ligates CXCR6 chemokine receptor, and guides migration of activated Th1 and Tc1 cells. It is identical to scavenger receptor SR-PSOX, which mediates uptake of oxidized low-density lipoprotein. We investigated whether CXCL16 expression is controlled by interferon-gamma (IFN-gamma)-cytokine abundant in atherosclerotic lesions., Methods and Results: CXCL16 and CXCR6 expression was identified by polymerase chain reaction and histochemistry in atherosclerotic lesions from humans and apolipoprotein-E-deficient mice. In vitro IFN-gamma induced CXCL16 in human monocytic THP-1 cells and primary human monocytes, which led to increased uptake of oxidized low-density lipoprotein in THP-1 cells, which could be blocked by peptide antibodies against CXCL16. In vivo IFN-gamma induced CXCL16 expression in murine atherosclerotic lesions., Conclusions: We demonstrate a novel role of IFN-gamma in foam cell formation through upregulation of CXCL16/SR-PSOX. CXCR6 expression in the plaque confirms the presence of cells able to respond to CXCL16. Therefore, this chemokine/scavenger receptor could serve as a molecular link between lipid metabolism and immune activity in the atherosclerotic lesion.

Atherosclerosis is an inflammatory disease. Both innate and adaptive immunity are involved in lesion formation and development. A number of antigen candidates, such as oxidized low-density lipoprotein and heat shock protein, have been associated with the inflammation and immune reaction that is part of the atherosclerotic process. Because experimental models of some other inflammatory/autoimmune diseases can be improved by vaccination, it is of interest to investigate if vaccination can also be applied to prevent or retard atherosclerosis. Indeed, the modification of immune responses in animal models can greatly affect the development and progression of atherosclerosis. This review provides an overview of our current understanding of effects and proposed mechanisms of immunization on preventing atherosclerosis.

Increasing evidence suggests that atherosclerosis is an inflammatory disease promoted by hypercholesterolemia. The role of adaptive immunity has been controversial, however. We hypothesized that proatherogenic T cells are controlled by immunoregulatory cytokines. Among them, TGF-beta has been implied in atherosclerosis, but its mechanism of action remains unclear. We crossed atherosclerosis-prone ApoE-knockout mice with transgenic mice carrying a dominant negative TGF-beta receptor II in T cells. The ApoE-knockout mice with disrupted TGF-beta signaling in T cells exhibited a sixfold increase in aortic lesion surface area, a threefold increase in aortic root lesion size, and a 125-fold increase in aortic IFN-gamma mRNA when compared with age-matched ApoE-knockout littermates. When comparing size-matched lesions, those of mice with T cell-specific blockade of TGF-beta signaling displayed increased T cells, activated macrophages, and reduced collagen, consistent with a more vulnerable phenotype. Ab's to oxidized LDL, circulating T cell cytokines, and spleen T cell activity were all increased in ApoE-knockout mice with dominant negative TGF-beta receptors in T cells. Taken together, these results show that abrogation of TGF-beta signaling in T cells increases atherosclerosis and suggest that TGF-beta reduces atherosclerosis by dampening T cell activation. Inhibition of T cell activation may therefore represent a strategy for antiatherosclerotic therapy.

Purpose: To investigate the potential role of caspase-3 in blue light-induced apoptosis in photoreceptor cells., Methods: Cyclic light-raised Sprague-Dawley rats were exposed to 400 to 480 nm light for 6 hours at an irradiance of 0.64 W/m(2). The rats were then kept in darkness for recovery and killed at scheduled time points from 0 hours up to 24 hours after exposure to light. The unexposed rats were used as the control. Apoptosis was marked with in situ terminal dUTP nick end labeling (TUNEL). Caspase-3 expression was explored using immunohistochemistry, Western blot analysis, real-time RT-PCR, and enzyme activity assay., Results: Exposure to blue light resulted in photoreceptor cell apoptosis, mostly in the superior temporal area of the retina. TUNEL-positive cells were highest during 8 to 16 hours of recovery in darkness. Procaspase-3 protein was constitutively expressed in the rat retina and was apparently upregulated after exposure to light, with expression peaking at 8 to 16 hours and subsiding at 24 hours. The upregulation was further supported by enhanced transcription of caspase-3 in the postexposure retina. Meanwhile, increased cleavage of caspase-3 into its active fragments of 17 and 19 kDa was detected after exposure to light, peaking at 16 hours. Activation of caspase-3 was subsequently located in the photoreceptor cells and predominantly in the superior part of the temporal quadrant. This coincided with elevated caspase-3-like activity at 16 hours after exposure to light., Conclusions: Procaspase-3 protein is temporally upregulated in the retina after in vivo exposure to blue light, and the upregulation is coupled to activation of caspase-3 and concomitant induction of apoptosis in the photoreceptor cells.