Atherosclerotic Abdominal Aortic Aneurysm and the Interaction Between Autologous Human Plaque-Derived Vascular Smooth Muscle Cells, Type 1 N
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循环研究杂志 2005年第4期
the Department of Biochemical Pharmacology (W.L.C., N.P., T.V.L., D.P.), William Harvey Research Institute, Queen Mary, University of London, London, UK
General Surgery (H.H., S.M.K.), Barnet and Chase Farm Hospital, Enfield, UK
the Laboratory of Immunology (A.P.), National Institute for Cancer Research, Genoa, Italy.
Abstract
Immune cell infiltration, vascular smooth muscle cell (VSMC) proliferation, and apoptosis are pathological hallmarks of atherosclerosis. The multifocal, chronic, and inflammatory nature of this disease of the cardiovascular system complicates targeted cellular therapy and emphasizes the need to understand the role and interaction of immune cells with VSMCs. We characterized the immune cell subsets present in human atherosclerotic tissue derived from atherosclerotic abdominal aortic aneurysm (AAA) and expanded them to study their interaction with autologous plaque-derived VSMCs in vitro. We show here that apart from T lymphocytes, plaque infiltrates consist of lots of NK cells and significant proportions of NKT cells that express T cell receptor (TCR) , CD4, and the NK markers CD56 and CD161. The infiltrates are predominantly IFN-eCproducing Type 1 lymphoid cells. When cocultured, the T and NKT cells adhere to VSMCs. CD4+ T cells enhance VSMC proliferation. VSMCs in turn enhance CD4+CD161+ NKT but not CD4+ or CD8+ T cell proliferation. CD4+CD161+ NKT cells inhibit VSMC proliferation by inducing apoptosis. Our results suggest that the interactions of Type 1 CD4+ T and CD4+CD161+ NKT cells with VSMCs may regulate VSMC proliferation and death respectively in atherosclerosis and the balance of these interactions could determine plaque stability.
Key Words: atherosclerotic abdominal aortic aneurysm helper T cells NKT vascular smooth muscle cells atherosclerosis
Introduction
Atherosclerosis is an important chronic inflammatory disease of the vascular system.1,2 It is a slow dynamic progressive disease characterized by atherosclerotic lesions composed of dysfunctional endothelium, smooth muscle cells, lipid-laden macrophages, and T lymphocytes within walls of large elastic and muscular arteries.3 In addition to the immune response,4 the migration of vascular smooth muscle cells (VSMCs) into the intima of arterial walls and their focused proliferation in the intima is a key process of atherogenesis.5 VSMCs can also undergo apoptosis, resulting in plaque weakness.6,7 These processes can lead to a number of different vascular phenomena such as positive remodeling, vessel stenosis, aneurysm formation, or acute plaque rupture leading to thrombotic vessel occlusion. Atherosclerotic aneurysm, the common form of fusiform infrarenal human abdominal aortic aneurysms (AAAs), is usually accompanied by and developed from atherosclerotic plaques. The most prevalent features of atherosclerotic AAA include apoptosis of VSMCs and intense inflammatory cell (T lymphocytes and macrophages) infiltration of the aortic wall and increased cytokine and protease activity.8,9 Thus there is an imperative to understand the role and interaction of immune cells with VSMCs to allow targeted cellular therapy in this disease.
T lymphocytes, present in lesions at all stages of the disease, are a fundamental component of the histopathology of early and late lesions of atherosclerosis.1 They are more than an accidental bystander as many of these T cells express activation markers.10 In addition, there is evidence that plaque T cells are actively dividing11 and T cell cytokines like IFN- are expressed within atherosclerotic lesions of both human and mice.10,12 However, the types and distribution of T cell responses, and the mechanism by which they influence the disease process, are still not clear. Although FasL/Fas-mediated apoptosis of plaque-derived VSMCs have been investigated, such studies were conducted with monocytes derived from peripheral blood,13 with limited reflection on the cellular microenvironment of the plaque. Thus no studies have so far been performed to directly investigate the interaction of VSMCs with lymphoid cells (T and NKT), all plaque-derived. Such studies could be important in view that IFN- has been shown to potentiate Fas-mediated apoptosis14 and IFN- is a prominent proinflammatory mediator of atherosclerosis as evidenced by recent in vitro and in vivo studies that demonstrated that it could promote atherosclerosis.15,16 Whereas IFN- is largely produced by T lymphocytes, NK, and NKT cells,17eC19 the precise role of these lymphoid cells, and what triggers their activation to IFN- production and subsequent proatherogenic activity, is unclear.
NKT cells represent a subset of mature unconventional T cells that is defined as expressing both the T cell receptor (TCR) and markers characteristic of NK cells such as CD161 (NK1.1), CD122 (IL-2R), and CD5620eC22 and have distinctive phenotypic and functional properties.19,23 They have been extensively characterized in mice but are also present in human, and differences have been reported between the two species. The prototypic NKT cells express the invariant TCR chain (V24JQ in the human) and are restricted by the nonpolymorphic class 1 molecule, CD1d. They are mainly of the CD4+ or CD4eCCD8eC phenotype and can be detected wherever conventional T cells are found, although the proportion varies widely in a tissue-specific manner. Functionally, NKT cells exert potent cytolytic activity mediated by perforin/granzyme B and FasL.24 Both mouse and human NKT cells rapidly secrete cytokines associated with type 1 (IFN-, IL-2, TNF-) or type 2 (IL-4, IL-5, IL-10, IL-13) responses on TCR engagement and reaction with glycolipid antigen.19,23 This apparent Th0 profile of cytokine production may be skewed toward a predominance of IFN-25 or IL-426 to promote Type 1 responses under certain conditions and Type 2 responses in others, respectively. This coincides with their stable expression of IL-18R or ST2L, which are Type 1 and Type 2 markers, respectively.27eC29 Although NKT cells are not required for the generation of polarized helper T (Th) cell response, the type of responses ascribed to NKT range from immunosuppression to immunoaggression.22 Furthermore, it has been recently reported that activation of CD1d-restricted NKT cells exacerbates atherosclerosis in vivo.30 However, their mechanistic role is still not clear.
Throughout this study, we used cultures of VSMCs and lymphoid cells (both T and NKT) that were established from AAA tissues rich in atherosclerotic plaques from patients requiring surgery for atherosclerotic aneurysm, expanded in vitro, and characterized by staining for cell-specific markers. In this report, we investigated the nature and composition of the lymphoid cell infiltrates in atherosclerotic plaques and examined their interaction with plaque-derived autologous VSMCs using in vitro experiments. The present results show for the first time, not only the presence of type 1 NK and TCR+CD4+CD161+NKT cells in plaques, but also that they and CD4+ Th cells, have specific interaction with VSMCs. Such interactions, if unbalanced, may culminate in vascular inflammation that contributes to the pathobiology of atherosclerosis.
Materials and Methods
Patient Specimens
Atherosclerotic tissues were obtained from patients with abdominal aneurysm undergoing elective surgery for graft repair of the infrarenal abdominal aorta. Fifteen patients, 12 men and 3 women, aged between 55 to 85 years old were included in the study. They are all vasculopaths with other features of atherosclerosis such as myocardial ischemia, peripheral vascular disease, cerebrovascular disease, or renal atherosclerotic impairment, suggesting an atherosclerotic as opposed to vasculitic or connective tissue etiology. The patients’ demographic profile is presented in online Table I (see the online data supplement). Most of the patients have at least three risk factors for atherosclerosis and are hypertensive, smokers, and male but not diabetic. The tissues were removed from the aortic sacs at the time of surgery, placed immediately in chilled RPMI 1640 medium, and processed within 24 hours. All the tissues were obtained with patient consent and local ethical approval.
For an expanded Materials and Methods section, see the online data supplement available at http://circres.ahajournals.org.
Results
Presence of Immune Infiltrates in Atherosclerotic Plaques in AAA
To demonstrate the presence of atherosclerotic plaques and to locate inflammatory cells in human AAA tissue, consecutive cryosections were stained with the histological stains hematoxylin and eosin, Verhoeff von Giessen, or Oil red O with hematoxylin (Figure 1a). We demonstrate in this study a section of aneurysmal tissue that contains an atherosclerotic plaque with a lipid core stained with oil red O (top right), and this is evident in all 15 patients. Furthermore, clumps of lymphoid infiltrates are found mainly in the media and intima surrounding the plaque and not in the adventitia (top left), suggesting that the lymphoid cells are involved with the atherosclerotic process. This is typical of other atherosclerotic AAA tissue sections tested. Characteristic of aneurysm tissue, there is increased neovascularization with reduced elastin in the media and increased collagen mass in the adventitia (bottom). The presence of immune infiltrates in the plaque and media is further supported by 3-color immunohistochemistry of this and other consecutive cryosections of the same tissue (Figure 1b). In the tissue section shown, there is an abundance of TCReCCD161+ NK (red cells, bottom left) and TCR+CD161+ NKT cells (yellowish green/ red cells, bottom left) in this clump of infiltrates near a region of neovascularization, and they are type 1 IFN-eCproducing cells (bottom right) as evidenced by their expression of IL-18R, a type 1 marker.27,29 These results indicate that apart from T lymphocytes, NK and NKT cells are present in very high proportions in atherosclerotic AAA tissue.
Predominance of Type 1 T, NK, and NKT Cells in Plaques
We have recently demonstrated that type 1 (IFN- producing) lymphoid cells stably express IL-18R, whereas type 2 (IL-4 producing) lymphoid cells stably express ST2L in mice and human.27eC29,31 To assess the proportion of type 1 and type 2 lymphoid cells in the infiltrates of plaques, we performed 3-color flow cytometric analysis on ex vivo immune cells from 15 AAA tissues using antibodies to IL-18R or ST2L with antibodies to appropriate combinations of the T cell markers TCR, V24, CD3, or CD4 and the NK cell markers CD161 or CD56. Our data not only further support the presence of NK and NKT cells in immune infiltrates within human atherosclerotic aneurysmal tissue, but that there are higher proportions of type 1 CD4+ T helper (Th), NK, as well as the TCR+CD56+/CD161+, CD3+CD56+/CD161+, and V24+CD56+/CD161+ NKT cells, compared with type 2 cells, in human atherosclerotic plaques (Figure 2). Being predominantly IL-18R+, these type 1 lymphoid cells are chiefly responsible for elaborating the central proatherogenic factor, IFN-.27,29 The proportion of invariant V24+ NKT is variable between tissues and may constitute only 15% of total NKT. Furthermore, the proportion of different lymphoid cell subsets (T, NK, and NKT) can vary depending on the location of the infiltrate.
VSMCs Support NKT but not T Cell Growth
To test our hypothesis for the involvement of NKT cells in atherosclerosis, plaque lymphoid cells (both T and NKT) were initially expanded in vitro with immobilized anti-CD3 and anti-CD161 or anti-CD28 as costimulators in the presence of autologous plaque VSMCs. The lymphoid cells and VSMCs proliferated well and expansion was enhanced by direct contact between lymphoid cells and VSMCs in the culture, as shown by large clumps of blasting lymphoid cells adhering tightly to VSMC fibers (Figure 3a). The observed lymphoid cell activation and proliferation was not due to allogenic response because autologous VSMCs and lymphoid cells were used. Adherence of both T and NKT cells was confirmed in cocultures of VSMCs with lymphoid cells and by 3-color immunostaining, which shows one blue-colored CD3+CD161eC T cell and a reddish blue CD3+CD161+ NKT cell adhering to the green -actin containing VSMCs (Figure 3b through 3g). To further investigate the nature of the interaction between VSMCs and T or NKT cells, the lymphoid cells were further expanded with restimulation in the absence of VSMCs before positive selection for NKT and T cells. Before positive selection, the lymphoid cells were screened to estimate the proportion of CD4+CD56+/CD161+TCR+ NKT cells available for selection. Because 65% of CD161+TCR+ NKT and all CD161eCTCR+ T cells express CD4 (Figure 4a) and none of the CD4+CD161+ NKT cells express a TCR (Figure 4b), the lymphoid cells were then positively sorted into CD4+ T or CD4+CD56+/CD161+ NKT cells and confirmed by flow cytometry before use. They were predominantly type 1 CD3+CD161+IL-18R+ NKT or CD3+CD161eCIL-18R+ CD4+ Th cells (Figure 4c and 4d), respectively. VSMCs support of NKT cell growth was confirmed by monitoring, via 3H-thymidine incorporation, the proliferation of positively sorted CD3+CD4+CD161+ NKT cells in the absence or presence of fixed autologous VSMCs (Figure 5a). Such enhancement of NKT cell proliferation was dependent on direct contact with fixed VSMCs, as the effect was abrogated by pretreatment of fixed VSMCs with anti-VCAM or anti-CD40 but not with anti-ICAM or control normal mouse IgG. We next investigated whether, apart from direct contact, human VSMCs could also produce IL-15, an important growth-promoting cytokine for NKT cells.32 In the presence of IFN-, VSMCs are induced to produce substantial amounts of IL-15 (Figure 5b). Therefore, as a proatherogenic factor, IFN- activates VSMCs to produce IL-15 and also induces the expression of VCAM,33 a molecule important in the induction of NKT cell proliferation by VSMCs (Figure 5a). In contrast, contact with fixed VSMCs had no effect on CD4+ (Figure 5c) or CD8+ T cell (data not shown) proliferation. These results indicate that unlike conventional T cells, innate T cells (NKT) can be induced, via ligation of VLA-4 and CD40L on their surface, to proliferate in the presence of IL-15 produced by VSMCs.
CD4+ T Cells Induce VSMC Proliferation Whereas NKT Cells Induce VSMC Apoptosis
Next, we investigated the effect of positively selected CD4+ T or NKT cells on VSMC proliferation by direct cell-cell contact. Fixed CD4+ T cells enhanced autologous VSMC proliferation on contact (Figure 6a). The mechanism involved is currently not clear as the proliferation is not affected by blocking VCAM-1, ICAM-1, or CD40 ligation with T cells that were pretreated with antieCVLA-4, antieCLFA-1, and anti-CD40L antibody. Interestingly, this process was contrasted by our demonstration in this study that NKT cells induced autologous VSMCs to apoptose. This was demonstrated by the inhibition of VSMC proliferation in the presence of fixed NKT cells (Figure 6b). The inhibition was almost completely reversed by the presence of the broad-spectrum caspase inhibitor z-VAD-FMK, suggesting that NKT cells were inducing VSMC apoptosis. The induction of apoptosis in VSMCs was further confirmed by staining VSMCs, treated or not with fixed NKT cells, with annexin V-FITC and propidium iodide. In the presence z-YVAD-FMK, the inhibitor specific for caspase 1/ICE and caspase 4, the reversal (40%) of apoptosis was, however, less dramatic (Figure 6c). This suggests that some other caspase(s) may also be involved in the apoptotic process. In addition, when VSMCs were cultured in the presence of IFN- or spent NKT culture medium (SN) containing 788pg/mL IFN-, they showed a substantial increase (50%) in expression of Fas compared with in the presence of medium (med) alone (Figure 6d), indicating that IFN- produced by NKT1 cells could upregulate Fas expression on VSMCs. This could synergistically promote FasL-mediated apoptosis inducible by NKT cells. These results indicate that T and NKT differ functionally in their interaction with VSMCs.
Discussion
In this study, we show for the first time that apart from T cells, plaque-immune infiltrates in all 15 atherosclerotic AAA tissues examined contain a significant proportion of NK and TCR+CD161+ NKT cells, and this varies widely between individuals and may depend on the disease activity, consistent with the marked heterogeneity of AAA in general. Normal aorta does not contain inflammatory infiltrates and contains few immune cells.34,35 Because we are not studying the tissue distribution and density of these inflammatory cells with disease progression, a normal aorta was not used. The Th, NK, and NKT cells are strongly positive for the IL-18R chain and therefore are predominantly type 1 lymphoid cells that produce IFN-. Among our expanded lymphoid cells, up to 65% of TCR+CD161+ NKT cells express CD4 and no CD4+CD161+ NKT cells are TCR positive. Because of this, we used the CD4+CD161+TCR+ NKT cell subset to focus our study of the interaction of NKT cells with VSMCs. Hence, when plaque-derived NKT, T, and VSMCs are expanded in vitro and cocultured, CD4+ Th cells induce VSMC proliferation. VSMCs induce NKT but not CD4+ or CD8+ T cell proliferation. NKT but not CD4+ Th cells induce VSMC apoptosis. These results suggest a mechanism by which CD4+ Th and NKT cells may regulate VSMC proliferation and death, respectively, a pathological hallmark of atherosclerosis. This could have significant in vivo implications because apoptosis of VSMCs has recently been identified as a key feature in plaque stability in atherosclerosis and is a major determinant of VSMC number in physiological remodeling of the vasculature.36
In mice, NKT cells have suppressor functions important in immunoregulation and prevention of autoimmunity in mice.37 However, unlike other T-regulatory cells, they can also act as aggressors in immune responses to some pathogens37 and as suppressors in anti-tumor immunity.22 This dual function of NKT cells is attributed to their ability to rapidly secrete cytokines associated with type 1 (IFN-) or type 2 (IL-4) responses on TCR engagement and reaction with glycolipid antigen.19,23 Cytokines such as IL-12 and IL-18 can also stimulate NKT cells to release IFN- and exhibit natural cytotoxicity.38 Furthermore, on activation, NKT cells also exert potent cytolytic activity mediated by perforin/granzyme B and FasL.22,24 In this study, we present data to show that NKT cells may be immunoaggressors in atherosclerosis. This is supported by our demonstration that NKT cells can inhibit VSMC growth by inducing them to apoptose via Fas/FasL interaction, a process that is mediated by ICE-like proteases/caspases39,40 and so is almost completely reversed by the broad spectrum caspase inhibitor z-VAD-FMK (Figure 6b). Furthermore, we show that IFN- produced by type 1 NKT (NKT1) cells in culture, can upregulate Fas expression on VSMCs (Figure 6d). This may synergistically promote the Fas/FasL-mediated VSMC apoptosis induced by NKT cells. As Fas/FasL-mediated apoptosis may be triggered in VSMCs at sites of high inflammatory content,36 our results suggest that NKT1 cells in plaque infiltrates may produce IFN- to sensitize VSMCs and induce apoptosis by Fas/FasL interaction. This could lead to plaque rupture when NKT1 cells are present in the shoulder region of plaques. In addition, NKT cells present in the media could also contribute to extracellular matrix degradation and pronounced decrease in medial VSMC density common in advanced AAA,41 as a result of VSMC apoptosis.
We have also demonstrated for the first time that there is strong CD40/CD40L interaction between NKT and VSMCs (Figure 5a), showing that VSMCs can induce NKT cell proliferation via CD40/CD40L interaction. In the context that NKT cells in turn can induce VSMC apoptosis, this may act as a negative feedback mechanism to reduce exuberant VSMC proliferation, or this may be a property of the VSMCs that have been isolated from the particular microenvironment of the aneurysm, where loss of VSMCs and hence vessel wall weakness is a key part of the pathophysiology. We also note that CD40 ligation activates Caspase1/ICE activity as well as the production of matrix-degrading enzymes in VSMCs,42,43 both of which can lead to apoptosis.36 Although NKT1 can induce apoptosis in VSMCs via Fas/FasL interaction, other stimuli and pathways exist that may act synergistically with Fas. This is demonstrated in Figure 6c where z-YVAD-FMK, a selective inhibitor of Caspase 1 can only partially reverse (40%) the VSMC apoptosis induced via CD40 ligation by fixed NKT1, leaving the remaining concurrent Fas-mediated apoptosis, where caspase 8 is an essential and apical initiator of the Fas signaling pathway,44,45 and other effector caspases unaffected. Although caspase-1 is involved in Fas-mediated apoptosis of thymocytes, it does not play a requisite role.46 Therefore, the partial inhibitory effect of z-YVAD-FMK on ICE may reflect synergistic effect of the Fas and CD40 signaling pathways in NKT-induced VSMC apoptosis. Furthermore, NKT1 can also kill via granzyme B/perforineCmediated pathway24 although that pathway is not included in the present study using fixed NKT cells.
By contrast, CD4+ Th1 cells may enhance plaque stability by inducing VSMC proliferation (Figure 6a) through unknown mechanisms. Therefore, we show here for the first time the different and opposing mechanistic roles of the innate (NKT) and adaptive (CD4+ T) lymphocytes isolated from atherosclerotic tissues. Because of their opposing functions, their balance in the plaque may determine its stability or susceptibility to rupture. In this context, it is not surprising that whereas Th1 cells also produce IFN-, although much less to sensitize Fas and possibly lower FasL expression, the net effect is insufficient to induce significant apoptosis in VSMCs.
In conclusion, our in vitro analysis of the interaction between VSMCs and Th1 or NKT1 cells within atherosclerotic tissue reveals further complexity in atherosclerosis. We demonstrate for the first time, not only the presence of type 1 NKT cells in plaques, in addition, we propose a mechanism by which they and CD4+ Th1 cells may regulate VSMC proliferation or apoptosis. CD4+ Th1 lymphocytes, via their ability to enhance VSMC proliferation, may help to maintain plaque stability through the formation of a firm fibrous cap. This may be followed by enhanced expansion of NKT1 cells produced by the increased presence of VSMCs. NKT1 cells, in turn via Fas, IFN- production, and CD40 signaling, may induce VSMC apoptosis. With time, a disproportionate accumulation of NKT1 cells in conjunction with appropriate costimulation may culminate in plaque rupture as a result of VSMC apoptosis induced by NKT1 cells. The increased presence and destabilizing role of NKT cells in plaques may also provide an explanation for the clinical observation that by lowering lipid levels, statins reduce acute adverse coronary events, and this correlates with diminished vascular inflammation and activation47,48 as it has been shown that glycolipids such as the synthetic glycolipid -galactosylceramide are required for NKT cell stimulation49 and atherosclerotic lesions are known to have higher concentrations of glycolipids than unaffected aortas. The balance of acquired (T cells) and innate (NKT cells) immunity may be important in atherosclerotic disease regulation. This may be determined by the extent of CD40L signaling, required for the induction of NKT cell proliferation by VSMCs (Figure 5a), and the effect may explain the reduction of atherosclerosis seen on administration of neutralizing anti-CD40L antibody in mice.50
This study has not only provided us with new insights into the pathogenesis of lesion progression, but could lead to novel therapeutic approaches involving immune modulation. Specifically, through understanding the proatherogenic role of NKT cells, it could provide a better understanding of how to manipulate plaque stability and further reduce the acute thrombotic complications of this disease.
Acknowledgments
This work was supported by the British Heart Foundation and The Wellcome Trust.
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Kawano T, Cui J, Koezuka Y, Toura I, Kaneko Y, Motoki K, Ueno H, Nakagawa R, Sato H, Kondo E, Koseki H, Taniguchi M. CD1d-restricted and TCR-mediated activation of valpha14 NKT cells by glycosylceramides. Science. 1997; 278: 1626eC1629.
Mach F, Schonbeck U, Sukhova GK, Atkinson E, Libby P. Reduction of atherosclerosis in mice by inhibition of CD40 signalling. Nature. 1998; 394: 200eC203.(Woon Ling Chan, Nada Pejn)
General Surgery (H.H., S.M.K.), Barnet and Chase Farm Hospital, Enfield, UK
the Laboratory of Immunology (A.P.), National Institute for Cancer Research, Genoa, Italy.
Abstract
Immune cell infiltration, vascular smooth muscle cell (VSMC) proliferation, and apoptosis are pathological hallmarks of atherosclerosis. The multifocal, chronic, and inflammatory nature of this disease of the cardiovascular system complicates targeted cellular therapy and emphasizes the need to understand the role and interaction of immune cells with VSMCs. We characterized the immune cell subsets present in human atherosclerotic tissue derived from atherosclerotic abdominal aortic aneurysm (AAA) and expanded them to study their interaction with autologous plaque-derived VSMCs in vitro. We show here that apart from T lymphocytes, plaque infiltrates consist of lots of NK cells and significant proportions of NKT cells that express T cell receptor (TCR) , CD4, and the NK markers CD56 and CD161. The infiltrates are predominantly IFN-eCproducing Type 1 lymphoid cells. When cocultured, the T and NKT cells adhere to VSMCs. CD4+ T cells enhance VSMC proliferation. VSMCs in turn enhance CD4+CD161+ NKT but not CD4+ or CD8+ T cell proliferation. CD4+CD161+ NKT cells inhibit VSMC proliferation by inducing apoptosis. Our results suggest that the interactions of Type 1 CD4+ T and CD4+CD161+ NKT cells with VSMCs may regulate VSMC proliferation and death respectively in atherosclerosis and the balance of these interactions could determine plaque stability.
Key Words: atherosclerotic abdominal aortic aneurysm helper T cells NKT vascular smooth muscle cells atherosclerosis
Introduction
Atherosclerosis is an important chronic inflammatory disease of the vascular system.1,2 It is a slow dynamic progressive disease characterized by atherosclerotic lesions composed of dysfunctional endothelium, smooth muscle cells, lipid-laden macrophages, and T lymphocytes within walls of large elastic and muscular arteries.3 In addition to the immune response,4 the migration of vascular smooth muscle cells (VSMCs) into the intima of arterial walls and their focused proliferation in the intima is a key process of atherogenesis.5 VSMCs can also undergo apoptosis, resulting in plaque weakness.6,7 These processes can lead to a number of different vascular phenomena such as positive remodeling, vessel stenosis, aneurysm formation, or acute plaque rupture leading to thrombotic vessel occlusion. Atherosclerotic aneurysm, the common form of fusiform infrarenal human abdominal aortic aneurysms (AAAs), is usually accompanied by and developed from atherosclerotic plaques. The most prevalent features of atherosclerotic AAA include apoptosis of VSMCs and intense inflammatory cell (T lymphocytes and macrophages) infiltration of the aortic wall and increased cytokine and protease activity.8,9 Thus there is an imperative to understand the role and interaction of immune cells with VSMCs to allow targeted cellular therapy in this disease.
T lymphocytes, present in lesions at all stages of the disease, are a fundamental component of the histopathology of early and late lesions of atherosclerosis.1 They are more than an accidental bystander as many of these T cells express activation markers.10 In addition, there is evidence that plaque T cells are actively dividing11 and T cell cytokines like IFN- are expressed within atherosclerotic lesions of both human and mice.10,12 However, the types and distribution of T cell responses, and the mechanism by which they influence the disease process, are still not clear. Although FasL/Fas-mediated apoptosis of plaque-derived VSMCs have been investigated, such studies were conducted with monocytes derived from peripheral blood,13 with limited reflection on the cellular microenvironment of the plaque. Thus no studies have so far been performed to directly investigate the interaction of VSMCs with lymphoid cells (T and NKT), all plaque-derived. Such studies could be important in view that IFN- has been shown to potentiate Fas-mediated apoptosis14 and IFN- is a prominent proinflammatory mediator of atherosclerosis as evidenced by recent in vitro and in vivo studies that demonstrated that it could promote atherosclerosis.15,16 Whereas IFN- is largely produced by T lymphocytes, NK, and NKT cells,17eC19 the precise role of these lymphoid cells, and what triggers their activation to IFN- production and subsequent proatherogenic activity, is unclear.
NKT cells represent a subset of mature unconventional T cells that is defined as expressing both the T cell receptor (TCR) and markers characteristic of NK cells such as CD161 (NK1.1), CD122 (IL-2R), and CD5620eC22 and have distinctive phenotypic and functional properties.19,23 They have been extensively characterized in mice but are also present in human, and differences have been reported between the two species. The prototypic NKT cells express the invariant TCR chain (V24JQ in the human) and are restricted by the nonpolymorphic class 1 molecule, CD1d. They are mainly of the CD4+ or CD4eCCD8eC phenotype and can be detected wherever conventional T cells are found, although the proportion varies widely in a tissue-specific manner. Functionally, NKT cells exert potent cytolytic activity mediated by perforin/granzyme B and FasL.24 Both mouse and human NKT cells rapidly secrete cytokines associated with type 1 (IFN-, IL-2, TNF-) or type 2 (IL-4, IL-5, IL-10, IL-13) responses on TCR engagement and reaction with glycolipid antigen.19,23 This apparent Th0 profile of cytokine production may be skewed toward a predominance of IFN-25 or IL-426 to promote Type 1 responses under certain conditions and Type 2 responses in others, respectively. This coincides with their stable expression of IL-18R or ST2L, which are Type 1 and Type 2 markers, respectively.27eC29 Although NKT cells are not required for the generation of polarized helper T (Th) cell response, the type of responses ascribed to NKT range from immunosuppression to immunoaggression.22 Furthermore, it has been recently reported that activation of CD1d-restricted NKT cells exacerbates atherosclerosis in vivo.30 However, their mechanistic role is still not clear.
Throughout this study, we used cultures of VSMCs and lymphoid cells (both T and NKT) that were established from AAA tissues rich in atherosclerotic plaques from patients requiring surgery for atherosclerotic aneurysm, expanded in vitro, and characterized by staining for cell-specific markers. In this report, we investigated the nature and composition of the lymphoid cell infiltrates in atherosclerotic plaques and examined their interaction with plaque-derived autologous VSMCs using in vitro experiments. The present results show for the first time, not only the presence of type 1 NK and TCR+CD4+CD161+NKT cells in plaques, but also that they and CD4+ Th cells, have specific interaction with VSMCs. Such interactions, if unbalanced, may culminate in vascular inflammation that contributes to the pathobiology of atherosclerosis.
Materials and Methods
Patient Specimens
Atherosclerotic tissues were obtained from patients with abdominal aneurysm undergoing elective surgery for graft repair of the infrarenal abdominal aorta. Fifteen patients, 12 men and 3 women, aged between 55 to 85 years old were included in the study. They are all vasculopaths with other features of atherosclerosis such as myocardial ischemia, peripheral vascular disease, cerebrovascular disease, or renal atherosclerotic impairment, suggesting an atherosclerotic as opposed to vasculitic or connective tissue etiology. The patients’ demographic profile is presented in online Table I (see the online data supplement). Most of the patients have at least three risk factors for atherosclerosis and are hypertensive, smokers, and male but not diabetic. The tissues were removed from the aortic sacs at the time of surgery, placed immediately in chilled RPMI 1640 medium, and processed within 24 hours. All the tissues were obtained with patient consent and local ethical approval.
For an expanded Materials and Methods section, see the online data supplement available at http://circres.ahajournals.org.
Results
Presence of Immune Infiltrates in Atherosclerotic Plaques in AAA
To demonstrate the presence of atherosclerotic plaques and to locate inflammatory cells in human AAA tissue, consecutive cryosections were stained with the histological stains hematoxylin and eosin, Verhoeff von Giessen, or Oil red O with hematoxylin (Figure 1a). We demonstrate in this study a section of aneurysmal tissue that contains an atherosclerotic plaque with a lipid core stained with oil red O (top right), and this is evident in all 15 patients. Furthermore, clumps of lymphoid infiltrates are found mainly in the media and intima surrounding the plaque and not in the adventitia (top left), suggesting that the lymphoid cells are involved with the atherosclerotic process. This is typical of other atherosclerotic AAA tissue sections tested. Characteristic of aneurysm tissue, there is increased neovascularization with reduced elastin in the media and increased collagen mass in the adventitia (bottom). The presence of immune infiltrates in the plaque and media is further supported by 3-color immunohistochemistry of this and other consecutive cryosections of the same tissue (Figure 1b). In the tissue section shown, there is an abundance of TCReCCD161+ NK (red cells, bottom left) and TCR+CD161+ NKT cells (yellowish green/ red cells, bottom left) in this clump of infiltrates near a region of neovascularization, and they are type 1 IFN-eCproducing cells (bottom right) as evidenced by their expression of IL-18R, a type 1 marker.27,29 These results indicate that apart from T lymphocytes, NK and NKT cells are present in very high proportions in atherosclerotic AAA tissue.
Predominance of Type 1 T, NK, and NKT Cells in Plaques
We have recently demonstrated that type 1 (IFN- producing) lymphoid cells stably express IL-18R, whereas type 2 (IL-4 producing) lymphoid cells stably express ST2L in mice and human.27eC29,31 To assess the proportion of type 1 and type 2 lymphoid cells in the infiltrates of plaques, we performed 3-color flow cytometric analysis on ex vivo immune cells from 15 AAA tissues using antibodies to IL-18R or ST2L with antibodies to appropriate combinations of the T cell markers TCR, V24, CD3, or CD4 and the NK cell markers CD161 or CD56. Our data not only further support the presence of NK and NKT cells in immune infiltrates within human atherosclerotic aneurysmal tissue, but that there are higher proportions of type 1 CD4+ T helper (Th), NK, as well as the TCR+CD56+/CD161+, CD3+CD56+/CD161+, and V24+CD56+/CD161+ NKT cells, compared with type 2 cells, in human atherosclerotic plaques (Figure 2). Being predominantly IL-18R+, these type 1 lymphoid cells are chiefly responsible for elaborating the central proatherogenic factor, IFN-.27,29 The proportion of invariant V24+ NKT is variable between tissues and may constitute only 15% of total NKT. Furthermore, the proportion of different lymphoid cell subsets (T, NK, and NKT) can vary depending on the location of the infiltrate.
VSMCs Support NKT but not T Cell Growth
To test our hypothesis for the involvement of NKT cells in atherosclerosis, plaque lymphoid cells (both T and NKT) were initially expanded in vitro with immobilized anti-CD3 and anti-CD161 or anti-CD28 as costimulators in the presence of autologous plaque VSMCs. The lymphoid cells and VSMCs proliferated well and expansion was enhanced by direct contact between lymphoid cells and VSMCs in the culture, as shown by large clumps of blasting lymphoid cells adhering tightly to VSMC fibers (Figure 3a). The observed lymphoid cell activation and proliferation was not due to allogenic response because autologous VSMCs and lymphoid cells were used. Adherence of both T and NKT cells was confirmed in cocultures of VSMCs with lymphoid cells and by 3-color immunostaining, which shows one blue-colored CD3+CD161eC T cell and a reddish blue CD3+CD161+ NKT cell adhering to the green -actin containing VSMCs (Figure 3b through 3g). To further investigate the nature of the interaction between VSMCs and T or NKT cells, the lymphoid cells were further expanded with restimulation in the absence of VSMCs before positive selection for NKT and T cells. Before positive selection, the lymphoid cells were screened to estimate the proportion of CD4+CD56+/CD161+TCR+ NKT cells available for selection. Because 65% of CD161+TCR+ NKT and all CD161eCTCR+ T cells express CD4 (Figure 4a) and none of the CD4+CD161+ NKT cells express a TCR (Figure 4b), the lymphoid cells were then positively sorted into CD4+ T or CD4+CD56+/CD161+ NKT cells and confirmed by flow cytometry before use. They were predominantly type 1 CD3+CD161+IL-18R+ NKT or CD3+CD161eCIL-18R+ CD4+ Th cells (Figure 4c and 4d), respectively. VSMCs support of NKT cell growth was confirmed by monitoring, via 3H-thymidine incorporation, the proliferation of positively sorted CD3+CD4+CD161+ NKT cells in the absence or presence of fixed autologous VSMCs (Figure 5a). Such enhancement of NKT cell proliferation was dependent on direct contact with fixed VSMCs, as the effect was abrogated by pretreatment of fixed VSMCs with anti-VCAM or anti-CD40 but not with anti-ICAM or control normal mouse IgG. We next investigated whether, apart from direct contact, human VSMCs could also produce IL-15, an important growth-promoting cytokine for NKT cells.32 In the presence of IFN-, VSMCs are induced to produce substantial amounts of IL-15 (Figure 5b). Therefore, as a proatherogenic factor, IFN- activates VSMCs to produce IL-15 and also induces the expression of VCAM,33 a molecule important in the induction of NKT cell proliferation by VSMCs (Figure 5a). In contrast, contact with fixed VSMCs had no effect on CD4+ (Figure 5c) or CD8+ T cell (data not shown) proliferation. These results indicate that unlike conventional T cells, innate T cells (NKT) can be induced, via ligation of VLA-4 and CD40L on their surface, to proliferate in the presence of IL-15 produced by VSMCs.
CD4+ T Cells Induce VSMC Proliferation Whereas NKT Cells Induce VSMC Apoptosis
Next, we investigated the effect of positively selected CD4+ T or NKT cells on VSMC proliferation by direct cell-cell contact. Fixed CD4+ T cells enhanced autologous VSMC proliferation on contact (Figure 6a). The mechanism involved is currently not clear as the proliferation is not affected by blocking VCAM-1, ICAM-1, or CD40 ligation with T cells that were pretreated with antieCVLA-4, antieCLFA-1, and anti-CD40L antibody. Interestingly, this process was contrasted by our demonstration in this study that NKT cells induced autologous VSMCs to apoptose. This was demonstrated by the inhibition of VSMC proliferation in the presence of fixed NKT cells (Figure 6b). The inhibition was almost completely reversed by the presence of the broad-spectrum caspase inhibitor z-VAD-FMK, suggesting that NKT cells were inducing VSMC apoptosis. The induction of apoptosis in VSMCs was further confirmed by staining VSMCs, treated or not with fixed NKT cells, with annexin V-FITC and propidium iodide. In the presence z-YVAD-FMK, the inhibitor specific for caspase 1/ICE and caspase 4, the reversal (40%) of apoptosis was, however, less dramatic (Figure 6c). This suggests that some other caspase(s) may also be involved in the apoptotic process. In addition, when VSMCs were cultured in the presence of IFN- or spent NKT culture medium (SN) containing 788pg/mL IFN-, they showed a substantial increase (50%) in expression of Fas compared with in the presence of medium (med) alone (Figure 6d), indicating that IFN- produced by NKT1 cells could upregulate Fas expression on VSMCs. This could synergistically promote FasL-mediated apoptosis inducible by NKT cells. These results indicate that T and NKT differ functionally in their interaction with VSMCs.
Discussion
In this study, we show for the first time that apart from T cells, plaque-immune infiltrates in all 15 atherosclerotic AAA tissues examined contain a significant proportion of NK and TCR+CD161+ NKT cells, and this varies widely between individuals and may depend on the disease activity, consistent with the marked heterogeneity of AAA in general. Normal aorta does not contain inflammatory infiltrates and contains few immune cells.34,35 Because we are not studying the tissue distribution and density of these inflammatory cells with disease progression, a normal aorta was not used. The Th, NK, and NKT cells are strongly positive for the IL-18R chain and therefore are predominantly type 1 lymphoid cells that produce IFN-. Among our expanded lymphoid cells, up to 65% of TCR+CD161+ NKT cells express CD4 and no CD4+CD161+ NKT cells are TCR positive. Because of this, we used the CD4+CD161+TCR+ NKT cell subset to focus our study of the interaction of NKT cells with VSMCs. Hence, when plaque-derived NKT, T, and VSMCs are expanded in vitro and cocultured, CD4+ Th cells induce VSMC proliferation. VSMCs induce NKT but not CD4+ or CD8+ T cell proliferation. NKT but not CD4+ Th cells induce VSMC apoptosis. These results suggest a mechanism by which CD4+ Th and NKT cells may regulate VSMC proliferation and death, respectively, a pathological hallmark of atherosclerosis. This could have significant in vivo implications because apoptosis of VSMCs has recently been identified as a key feature in plaque stability in atherosclerosis and is a major determinant of VSMC number in physiological remodeling of the vasculature.36
In mice, NKT cells have suppressor functions important in immunoregulation and prevention of autoimmunity in mice.37 However, unlike other T-regulatory cells, they can also act as aggressors in immune responses to some pathogens37 and as suppressors in anti-tumor immunity.22 This dual function of NKT cells is attributed to their ability to rapidly secrete cytokines associated with type 1 (IFN-) or type 2 (IL-4) responses on TCR engagement and reaction with glycolipid antigen.19,23 Cytokines such as IL-12 and IL-18 can also stimulate NKT cells to release IFN- and exhibit natural cytotoxicity.38 Furthermore, on activation, NKT cells also exert potent cytolytic activity mediated by perforin/granzyme B and FasL.22,24 In this study, we present data to show that NKT cells may be immunoaggressors in atherosclerosis. This is supported by our demonstration that NKT cells can inhibit VSMC growth by inducing them to apoptose via Fas/FasL interaction, a process that is mediated by ICE-like proteases/caspases39,40 and so is almost completely reversed by the broad spectrum caspase inhibitor z-VAD-FMK (Figure 6b). Furthermore, we show that IFN- produced by type 1 NKT (NKT1) cells in culture, can upregulate Fas expression on VSMCs (Figure 6d). This may synergistically promote the Fas/FasL-mediated VSMC apoptosis induced by NKT cells. As Fas/FasL-mediated apoptosis may be triggered in VSMCs at sites of high inflammatory content,36 our results suggest that NKT1 cells in plaque infiltrates may produce IFN- to sensitize VSMCs and induce apoptosis by Fas/FasL interaction. This could lead to plaque rupture when NKT1 cells are present in the shoulder region of plaques. In addition, NKT cells present in the media could also contribute to extracellular matrix degradation and pronounced decrease in medial VSMC density common in advanced AAA,41 as a result of VSMC apoptosis.
We have also demonstrated for the first time that there is strong CD40/CD40L interaction between NKT and VSMCs (Figure 5a), showing that VSMCs can induce NKT cell proliferation via CD40/CD40L interaction. In the context that NKT cells in turn can induce VSMC apoptosis, this may act as a negative feedback mechanism to reduce exuberant VSMC proliferation, or this may be a property of the VSMCs that have been isolated from the particular microenvironment of the aneurysm, where loss of VSMCs and hence vessel wall weakness is a key part of the pathophysiology. We also note that CD40 ligation activates Caspase1/ICE activity as well as the production of matrix-degrading enzymes in VSMCs,42,43 both of which can lead to apoptosis.36 Although NKT1 can induce apoptosis in VSMCs via Fas/FasL interaction, other stimuli and pathways exist that may act synergistically with Fas. This is demonstrated in Figure 6c where z-YVAD-FMK, a selective inhibitor of Caspase 1 can only partially reverse (40%) the VSMC apoptosis induced via CD40 ligation by fixed NKT1, leaving the remaining concurrent Fas-mediated apoptosis, where caspase 8 is an essential and apical initiator of the Fas signaling pathway,44,45 and other effector caspases unaffected. Although caspase-1 is involved in Fas-mediated apoptosis of thymocytes, it does not play a requisite role.46 Therefore, the partial inhibitory effect of z-YVAD-FMK on ICE may reflect synergistic effect of the Fas and CD40 signaling pathways in NKT-induced VSMC apoptosis. Furthermore, NKT1 can also kill via granzyme B/perforineCmediated pathway24 although that pathway is not included in the present study using fixed NKT cells.
By contrast, CD4+ Th1 cells may enhance plaque stability by inducing VSMC proliferation (Figure 6a) through unknown mechanisms. Therefore, we show here for the first time the different and opposing mechanistic roles of the innate (NKT) and adaptive (CD4+ T) lymphocytes isolated from atherosclerotic tissues. Because of their opposing functions, their balance in the plaque may determine its stability or susceptibility to rupture. In this context, it is not surprising that whereas Th1 cells also produce IFN-, although much less to sensitize Fas and possibly lower FasL expression, the net effect is insufficient to induce significant apoptosis in VSMCs.
In conclusion, our in vitro analysis of the interaction between VSMCs and Th1 or NKT1 cells within atherosclerotic tissue reveals further complexity in atherosclerosis. We demonstrate for the first time, not only the presence of type 1 NKT cells in plaques, in addition, we propose a mechanism by which they and CD4+ Th1 cells may regulate VSMC proliferation or apoptosis. CD4+ Th1 lymphocytes, via their ability to enhance VSMC proliferation, may help to maintain plaque stability through the formation of a firm fibrous cap. This may be followed by enhanced expansion of NKT1 cells produced by the increased presence of VSMCs. NKT1 cells, in turn via Fas, IFN- production, and CD40 signaling, may induce VSMC apoptosis. With time, a disproportionate accumulation of NKT1 cells in conjunction with appropriate costimulation may culminate in plaque rupture as a result of VSMC apoptosis induced by NKT1 cells. The increased presence and destabilizing role of NKT cells in plaques may also provide an explanation for the clinical observation that by lowering lipid levels, statins reduce acute adverse coronary events, and this correlates with diminished vascular inflammation and activation47,48 as it has been shown that glycolipids such as the synthetic glycolipid -galactosylceramide are required for NKT cell stimulation49 and atherosclerotic lesions are known to have higher concentrations of glycolipids than unaffected aortas. The balance of acquired (T cells) and innate (NKT cells) immunity may be important in atherosclerotic disease regulation. This may be determined by the extent of CD40L signaling, required for the induction of NKT cell proliferation by VSMCs (Figure 5a), and the effect may explain the reduction of atherosclerosis seen on administration of neutralizing anti-CD40L antibody in mice.50
This study has not only provided us with new insights into the pathogenesis of lesion progression, but could lead to novel therapeutic approaches involving immune modulation. Specifically, through understanding the proatherogenic role of NKT cells, it could provide a better understanding of how to manipulate plaque stability and further reduce the acute thrombotic complications of this disease.
Acknowledgments
This work was supported by the British Heart Foundation and The Wellcome Trust.
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