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402 Stewart C. Whitman and Tanya A. Ramsamy

(α-GalCer), which is derived from marine sponges. In addition to being used as a way to distinguish NKT cells from NK cells, these four selection criteria have allowed NKT cells to be themselves partitioned into four broad categories as outlined in Fig. 18.1.

The ability of one unique subset of T lymphocytes to interact with CD1d results from the expression of a highly biased, evolutionarily conserved, TCR consisting of an invariant α-chain (a Vα14 segment joined to a Jα18 segment) that preferentially binds to one of three β-chains (Vβ8.2, Vβ-7, and Vβ-2) [70, 71]. As such, these T lymphocytes are known as invariant NKT cells [72] or Vα14 NKT cells [73]. In C57BL/6 mice, this unique subset of T lymphocytes generally, but not consistently, expresses the NK1.1 receptor to varying degrees [69, 74–76]. Although the physiological substrate for this subset of T lymphocytes is still unclear, invariant NKT cells have a strong response to and are selectively activated by the exogenous substrate, α- GalCer, a marine sponge-derived glycosphingolipid [77], which binds specifically to CD1d [74, 78]. Furthermore, invariant Vα14 T cells do not express CD8, a marker characteristic of cytotoxic T lymphocytes, but instead are either CD4+, a marker characteristic helper T lymphocytes, or double negatives (DN) [75, 76]. Although Vα14 NKT cells are by far the more abundant NKT cells present, other, less common NKT cells have been identified.

Like the Vα14 NKT cells, the non-Vα14 NKT cells are also CD1d-depend- ent and can either be CD4+ or DN [79–81]. This additional class of NKT cells, which expresses either the Vα3.2-Jα9 or Vα8 α-chain and preferentially binds the Vβ8 β-chain, is less common than the Vα14 NKT cells in the murine model [81]. Yet another class of NKT cells are those that are CD1dindependent and include NKT cells that express the NK1.1 receptor, have diverse TCR can be CD8+, CD4+, or DN [82, 83] or those that express CD49B, an antigen present on a majority of the murine NK cells [84].

Once activated, all NKT cell populations have the capacity to exert immunoregulatory functions by releasing large quantities of T helper (Th) 1 or Th2 cytokines. The Th1 cytokines release by NKT cells upon activation include the proatherogenic cytokines interleukin (IL)-12 [85, 86] and IFN-γ [87–90], whereas the antiatherogenic cytokines [86] and IL-10 [91–94] are associated with the Th2 response. The release of cytokines from NKT cells initiates a cascade of events and causes the bystander activation of adjacent NK cells, B lymphocytes, and dendritic cells [95], as well as the activation of conventional CD4+ and CD8+ T lymphocytes [71, 96]. Although the natural ligands for CD1d-dependent NKT cells, which includes the Vα14 NKT cells, still remain to be identified, α-GalCer has been shown to be a potent and specific activator of NKT cells [74]. As such, evidence exists to suggest that NKT cells can modulate antitumoral [97–99] and antimicrobial immunity [100, 101] as well as a number of autoimmune diseases, including experimental autoimmune encephalomyelitis [102] (a model for multiple sclerosis) and type 1 diabetes [103, 104], and as reviewed by van der Vliet et al. [105].

Atherosclerosis is a chronic inflammatory disorder that is known to involve components of both the innate and adaptive immune systems [3, 5, 106, 107]. Given that NKT cells link the two arms of the immune system, it seems likely that they would also play a role in the pathogenesis of atherosclerosis. In fact, several studies have shown, using lipopolysaccharide or α-GalCer, that NKT cells are present in atherosclerotic lesions [15, 18, 108]. Furthermore, α-GalCer administration has recently been shown to specifically exacerbate atherosclerosis in Apoe–/– mice, a model predisposed to developing atherosclerotic lesions spontaneously, when compared to vehicle treated animals [16, 18], and those also deficient in CD1d [15]. Incidentally, CD1d has been detected in human atherosclerotic lesions [109] underscoring the probable involvement of NKT cells in the human disease process. The administration of α-GalCer to Apoe–/– mice has been shown to have dramatic effects on the local cytokine environment of the aortas with established atherosclerotic lesions by inducing the production of IFN-γ, IL-4, and IL-10 [16]. Others have observed an early burst of inflammatory cytokines, both Th1 (IFN-γ, tumor necrosis factor-α, IL-2) and Th2 (IL-4, IL-5) cytokines, in the sera of Apoe–/– [16]. The effect of α-GalCer on the development of atherosclerosis and the production of proatherogenic cytokines by NKT cells suggests that CD1d-dependent NKT cells might play a participatory role in the atherogenic process.

CD1d Null Mice

Animal models that combine genetic risks for atherosclerosis, such as the Apoe–/– or the Ldl-r−/− [90] mouse, with an altered immune system have been invaluable in demonstrating a link between atherosclerosis and immunity [3, 5]. Using these models, several groups have provided evidence to suggest that NKT cells are potentially proatherogenic [15–19]. The most commonly used model of NKT cells deficiency is the CD1d–/– mouse, which was first created by Mendiratta et al. [110]. In this model, the CD1d–/– mice lack the molecule necessary for normal development of the CD1d-dependent NKT cell and are therefore deficient in functional CD1d-dependent NKT cells. Recent studies have shown that deficiency of CD1d significantly slows the development of atherosclerosis in wild-type mice on an atherogenic diet [18] or in Apoe–/– mice whether they were fed a chow diet [15] or an atherosclerotic diet, which is a diet enriched in both cholesterol and fat [16]. The reduction in atherosclerotic lesion size ranged from 25% in double knockouts on a chow diet to 70% on an atherosclerotic diet. Similar observations were shown when CD1d–/– was put onto an Ldl-r–/– genetic background. These mice had 50% less lesions than the CD1d+/+ controls when on an atherosclerotic diet for 4 weeks [17]. However, the authors did note that this difference was lost when both groups were compared at 8 and 12 weeks.

Using the technique of bone marrow transplantation following lethal irradiation, Nakai et al. [18] showed that Ldl-r–/– mice reconstituted with CD1d–/–

404 Stewart C. Whitman and Tanya A. Ramsamy

bone marrow had significantly less atherosclerosis compared to lethally irradiated Ldl-r–/– mice reconstituted with wild-type bone marrow. These data suggest that replenishing the hematopoietic stem cells with bone marrow from mice that have CD1d-dependent NKT cells is sufficient to stimulate the development of early stage atherosclerotic lesions. These results, combined with those obtained using CD1d null mice, suggest that the absence of CD1dreactive NKT cells attenuates lesion formation in mice during the early fatty streak formation.

Jα18 Null Mice

The exclusive expression of the invariant Vα14/Vβ receptor on Vα14 NKT cells and the essential requirement of Vα14 expression for the development of Vα14 NKT cells was demonstrated in Vα14 NKT cell-deficient (Jα18–/–) mice [111]. Here, Cui et al. [111] showed that targeted deletion of the Jα18 gene caused a complete failure of the mice to develop Vα14 NKT cells, leaving other lymphoid lineages intact. This observation strongly suggests that the invariant Vα14 segment is indispensable for the generation of Vα14 NKT cells. Targeted deletion of the Jα18 gene resulting in the selective depletion of CD1d-dependent, Vα14 NKT cells without affecting the population of other NKT (including the other CD1d-dependent NKT cells), NK, and conventional T lymphocytes [73, 111]. By superimposing the Jα18 deficiency onto mice that are already susceptible to atherosclerosis by virtue of being Ldl-r–/–, Whitman et al. have shown that the loss of functionally active Vα14 NKT cells significantly slows the formation of lesions by 20% in both genders of mice [19]. As such, this study shows that a single population of NKT cells participates in the process of early-stage atherosclerotic lesion formation. Upon stimulation, Vα14 NKT cells have the capacity to exert immunoregulatory functions by releasing large amounts of inflammatory cytokines, including the proatherogenic cytokine IFN-γ [87–90]. This release of cytokines will, in turn, cause the activation of adjacent NK cells, B cells, CD4+ and CD8+ T lymphocytes [71, 112], as well as adjacent antigen-presenting cells [95]. This hierarchical form of activation may be one of the key mechanisms by which NKT cells promote atherosclerosis at the level of the vessel wall.

Concluding Remarks

Despite the ever-growing list of immune cells now identified within the developing atherosclerotic lesion of both humans and mice, this chapter has only focused on two unique populations of lymphocytes, namely NK cells and NKT cells. As noted in the introduction, macrophages and conventional T lymphocytes constitute the major cell types involved in the earlyand latter stages of this disease. However, despite the apparent underrepresentation of both cell types, NK cells and NKT cells have now been shown to have a

significant participatory role alongside these other more “prominent” classes of lymphocytes. As highlighted above, recent experimental evidence from our laboratory [13, 14, 19] and others [15–18] suggest that neither NK cells nor NKT cells cause the disease, but rather these immune cells play an important role in accelerating lesion development by modulating, via the elaboration of IFN-γ, the function of other more prominent immune cells (conventional T lymphocytes and macrophages) found within the developing atherosclerotic lesion. In this context, research from a number of laboratories strongly suggests that the proinflammatory cytokines IL-12 [85, 86] and IL-18 [113, 114] play a significant role in promoting atherosclerosis via the activation of NKT and NK cells, respectively.

Current in vivo models that combine genetic risks for atherosclerosis with an altered immune system have revealed to us the important modulatory role played by both NK and NKT cells. These studies have also highlighted the fact that the transgenic mouse models of atherosclerosis, although not a perfect substitute for human lesion pathology, are currently our best all-around model that affords us a significant degree of flexibility by allowing us access to a vast array of different transgenic and gene-targeted mice. As new advancements occur in mouse gene manipulation techniques, the use of the mouse will increase our ability to define these complicated mechanistic pathways, leaving us with the easier task of having to only confirm such pathways in our human atherosclerotic specimens.

Acknowledgments: S. C. Whitman is the recipient of a Great-West Life & London Life New Investigator Award from the Heart and Stroke Foundation of Canada and is supported by the Heart and Stroke Foundation of Ontario Grant NA-5086 and Canadian Institutes of Health Research Grants MOP53344 and GHS-60663. T. A. Ramsamy is the recipient of a Heart and Stroke Foundation of Canada Post-Doctoral Fellowship awarded to the Coronary Disease and Heart Failure Group at the University of Ottawa Heart Institute. The authors apologize to those investigators whose excellent contributions to the field of atherosclerosis and immunity research, which could not be included in this review due to space limitations.

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