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Section IV

Other Factors in Atherosclerosis and

the Associated Complications

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work synergistically to promote lesion development. One of the emerging components that drive the development of both earlyand late-stage atherosclerotic lesions has been shown to be the participation of both the innate and acquired immune systems [3, 4]. With this said, atherosclerotic lesions are characterized by a pronounced infiltration of leukocytes at all stages of disease progression [2]. In both humans and animal models of atherosclerosis, the most prominent cells that infiltrate evolving lesions are macrophages and T lymphocytes [5–7]. The ablation of either of these cell types reduces the extent of atherosclerosis in mice that were rendered susceptible to the disease by deficiency of either apolipoprotein E (Apoe–/–) or the LDL receptor (Ldl-r–/–) [8–12]. In addition to these major immune cell participants, a number of less prominent leukocyte populations that can modulate the atherogenic process are also involved. Although this chapter will focus on the participatory role of two “less prominent” immune components, namely natural killer (NK) cells [13, 14] and natural killer T (NKT) cells [15–19], it should be pointed out that the process of atherogenesis has been shown to be modulated by small numbers of other types of immune cells, such as B lymphocytes [20–22], mast cells [23], and dendritic cells [24], as reviewed in greater detail by VanderLaan and Reardon [4].

The Contributions of the Immune System to the Various Stages of Atherosclerosis

In the arteries of healthy children, preexisting mononuclear cell infiltrations have been identified at regions known to have a greater likelihood for later developing atherosclerosis [25, 26]. These sites are speculated to function as local immunosurveillance systems that monitor the bloodstream for potentially harmful endogenous and exogenous antigens [26, 27]. The morphological feature of early-stage lesions in both Apoe–/– [28–30] and Ldl-r–/– mice [31] are very similar to those found in humans [32–34]. These lesions consist of an abnormal accumulation of lipoproteins and an assembly of immune cells, consisting mainly of T lymphocytes and macrophages [7], and to a lesser extent, NK cells, B cells, mast cells, and dendritic cells [13, 20, 24, 35].

The accumulation of plasma-derived lipoproteins is considered a necessary event in initiating atherosclerosis [36]. In vitro studies have shown that lipoproteins are trapped by matrix components [37–39] and modified by oxidation [40] within the tunica media to a form that is chemotactic for monocytes [41]. Oxidized lipoproteins may also be chemotactic for other immune cells such as NK cells, NKT cells, T- and B lymphocytes, facilitating their recruitment to the vessel wall [42]. Within the intima, monocytes differentiate to macrophages and begin to clear the modified lipoproteins via their scavenger receptors [43]. This culminates in the generation of numerous cholesterol ester-enriched “foam” cells and form what is commonly termed a fatty streak.

Advanced-stage atherosclerotic lesions have an accumulation of extracellular lipid, known as the lipid core. Macrophages, macrophage-derived foam cells and lymphocytes are found densely concentrated along the periphery of the lipid core of these lesions [33, 44]. In advance-stage human atherosclerotic lesions, lymphocytes [44], mast cells [45], NK cells [46], and NKT cells [47] have all been identified in the regions bordering the shoulder of the lipid core and the fibrous cap, with NK and NKT cells representing 0.1% and 2%, respectively, of the total lymphocyte population in these regions [46, 47]. Fibrous plaques that contain a hematoma and/or thrombotic deposits are termed complicated lesions, and are well documented in Apoe–/– mice [31, 48–51], but less so in Ldl-r–/– mice [31].

NK Cells and their Role in Innate Immunity

NK cells represent a subset of bone marrow-derived lymphocytes distinguishable from T- and B lymphocytes by their morphology, phenotype, and their ability to kill aberrant cells without prior sensitization [52]. In mice, as in humans, NK cells constitute only 10–15% of peripheral blood lymphocytes, but this distribution increases to 45% in certain areas such as the liver, peritoneal cavity, and placenta [53–55]. NK cells play an important role in maintaining the integrity of the innate immune defenses, as their primary role is believed to be one of providing early defense against pathogens during the initial response period while the adaptive immune system is being activated [56]. Functionally, NK cells act as effectors, either directly through the process of cell-mediated cytotoxicity upon degranulation with subsequent perforin and granzyme release, or through cytokine production, the most prominent being interferon (IFN)-γ, which in turn can mediate the activation of other effector cells that are in close apposition.

NK Cells and Experimental Atherosclerosis

Although a direct participatory role of NK cells in the process of human atherogenesis has not yet been shown, detailed immunohistochemical analysis of human autopsy specimens has shown the presence of NK cells at all stages of atherosclerotic lesion development [26, 46]. NK cells have also been detected in the Ldl-r–/– mouse model of atherosclerosis, yet unlike human lesions, only early-stage lesions in these mice were found to stain positive for NK cells [13, 57]. Interestingly, in both human and mouse atherosclerotic lesions, NK cells were found to make up only a small fraction of the lymphocyte population present in these lesions; approximately 0.1–0.5% of the total lymphocytes.

Animal models that combine genetic risks for atherosclerosis with an altered immune system have been invaluable in demonstrating a link between atherosclerosis and immunity [3, 58]. The identification of the beige mutation

400 Stewart C. Whitman and Tanya A. Ramsamy

mouse and the creation of the Ly49A transgenic mouse, two mouse strains that exhibit partial and complete NK cell deficiency, respectively, has allowed for the creation of a similar animal model aimed at defining the true role of NK cells in atherosclerosis.

Beige Mutation Mice

NK cell function is decreased in mice having the beige mutation [59–61] and these mice have been used in two separate atherosclerosis studies, yet these have yielded different results. Beige mice fed a diet enriched in saturated fat, cholesterol, and cholate did not exhibit any change in atherosclerotic lesion formation [62]. However, when the beige defect was bred onto the Ldl-r–/– background, there was a modest, but statistically significant increase in lesion size [57]. The beige mouse has a very complex phenotype, and while NK cell activity is decreased in these mice, the defect is not complete [59–61] allowing for residual NK cell activity to persist. Furthermore, given the nature of the mutation in the beige mice, which involves a poorly characterized protein required for proper lysosomal trafficking [59], disturbances in cell populations that are distinct from that of NK cells may ultimately have been responsible for the antiatherogenic effect noted [57].

Ly49A Transgenic Mice

Recently, transgenic mice have been developed that have defective natural cytotoxicity and a selective deficiency in functional NK1.1+ CD3– cells, while maintaining functionally normal T- and B lymphocytes [63]. This phenotype was achieved by expressing the inhibitory major histocompatibility complex (MHC) class I specific receptor, Ly49A, under the control of the granzyme A promoter. Ly49A is present on all NK cells and is a C-type lectin-like receptor that recognizes the MHC class I ligands, H-2D(d) and D(k). Interactions of these ligands with Ly49A inhibits activation of NK cells, which provides the rationale for the absence of the functional cells in these transgenic mice [63].

The development of transgenic mice with selective deficiency in NK activity affords the ability to define the specific role of NK cells in the development of atherosclerosis. Using the Ly49A transgenic mouse, Whitman and colleagues have shown that the deficiency of functional NK cells in both Ldlr–/– [13] and Apoe–/– [14] mice results in a significant reduction in the development of early-stage atherosclerotic lesions. Interestingly, lesions in Apoe–/– mice that carry the Ly49A transgene were found to advance in both size and complexity, such that after these mice had been fed an atherosclerosis-promoting diet for 12 weeks, the protective effect of NK cell deficiency was greatly diminished in female mice and completely lost in male mice [14].

NKT Cells and their Role in Linking the Innate and Acquired Immune Systems

Almost 20 years ago, three independent laboratories published studies identifying a previously unknown subset of T lymphocytes [64–66]. Nevertheless, it was not until 1995 that Makino et al. [67] coined the term NKT cell to describe a heterogeneous subset of mouse T lymphocytes that share some characteristics with NK cells [68] and appear to provide a link between the adaptive and innate immune systems. Despite this, it quickly became apparent that this broad definition of an NKT cell was inadequate, since some NKT cells lack an NK cell receptor. Furthermore, most murine strains, with the exception of the commonly used C57BL/6 strain in atherosclerosis studies, completely lack the expression of the classical NK cell receptor, NK1.1 [69]. In the absence of a truly definitive marker ubiquitously expressed on all NKT cells, a number of alternative criteria have been used to define this class of lymphocyte as unique from that of NK cells. The four most often used criteria are: (i) the ability of NKT cells to show autoreactivity to the nonclassical MHC molecule CD1d; (ii) the expression of a specific T cell receptor (TCR) reservoir of the NKT cell; (iii) the presence of NK cells receptors; and (iv) the responsiveness of the cell to the synthetic CD1d ligand, α-galactosylceramide

T lymphocytes

Invariant vα14 NKT cells

NKT cells

CD1D-restricted

T cells

FIGURE 18.1. NKT cells are a subset of T lymphocytes that share many characteristics with both NK cell and T lymphocytes. NKT cells are best described as a heterogeneous cell population that can be partitioned based on their reactivity to CD1d. The CD1ddependent NKT cells can further be segregated based on their TCR reservoir. Vα14 NKT cells, the most abundant form of NKT cells in the mouse, are composed of an invariant α chain and one of three β chains. CD1d-dependent T lymphocytes, including Vα14 T cells, can either be T lymphocytes or NKT cells depending on their receptor repertoire. Adapted from Wilson and Delovitch [71].