BRIEF REPORT |
Co-accumulation of Dendritic Cells and Natural Killer T Cells within Rupture-prone Regions in Human Atherosclerotic Plaques
Surgical Professorial Unit, St. Vincent's Hospital, University of New South Wales, Sydney, New South Wales, Australia
Correspondence to: Dr. Yuri V. Bobryshev, Surgical Professorial Unit, Level 5, DeLacy Building, St. Vincent's Hospital, Sydney, Darlinghurst, NSW 2010 Australia. E-mail: ybobryshev{at}stvincents.com.au
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Summary |
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(J Histochem Cytochem 53:781785, 2005)
Key Words: arteries atherosclerosis dendritic cells NKT cells
ATHEROSCLEROSIS is an immune-inflammatory disease that involves components of the innate and acquired immune system (Hansson et al. 2002). We reported previously that cells from the family of antigen-presenting dendritic cells (DCs) reside in large arteries (Bobryshev and Lord 1995
,1998
). Vascular DCs exhibit ultrastructural characteristics typical of other DCs (Bobryshev and Lord 1995
; Bobryshev 2000
) and, like Langerhans' cells and interdigitating cells (Lotze and Thomson 2001
), vascular DCs express S100 protein, actin-bundling protein p55 (fascin), and CD1a (Bobryshev and Lord, 1995
,1998
), which are markers for their immunohistochemical identification (Bobryshev 2000
). Like other DCs (Lotze and Thomson 2001
), vascular DCs express HLA-DR and display intercellular adhesion molecule-1 (ICAM-1) (Bobryshev and Lord 1998
). In the non-diseased arterial wall, DCs are regularly located along the subendothelial layer in numbers similar to those of Langerhans' cells in the skin, namely, 2% to 5% (Bobryshev 2000
). Millonig and colleagues (2001)
reported that in some intimal areas, vascular DCs form networks and suggested that DC networks are involved in the screening of potentially harmful antigens in the arterial wall. In atherosclerotic lesions, the numbers of DCs increase markedly, with more than 90% of DCs accumulated in atherosclerotic plaque shoulders, which represent plaque rupture-prone regions (Bobryshev and Lord 1998
; Bobryshev 2000
; Yilmaz et al. 2004
). Yilmaz and colleagues (2004)
reported that up to 70% of DCs in the shoulders of vulnerable carotid plaques express CD83, a marker of DC activation.
Inflammatory infiltrates in atherosclerotic plaques contain activated T cells (Hansson et al. 2002). Antigen-specific T-cell activation is dependent on the interactions of T-cell receptors (TCR) with antigens presented by major histocompatibility complex (MHC) molecules (Hansson et al. 2002
). ICAM-1/lymphocyte function-associated antigen-1 and vascular cell adhesion molecule-1 (VCAM-1)/very late antigen-4 interactions are critical in T cell activation (Hansson et al. 2002
). In atherosclerotic lesions, DCs display HLA-DR, ICAM-1, VCAM-1, and heat shock protein-70 and locate among and contact T cells (Bobryshev and Lord 1998
,2002
), suggesting that DCs might activate T cells directly in the injured arterial wall. CD1d, an antigen-presenting molecule responsible for the presentation of lipid antigens (Porcelli 1995
), is expressed in atherosclerotic lesions, and its expression is restricted to DCs (Bobryshev and Lord, 2002
). We hypothesized that CD1-restricted responses may play a role in atherosclerosis (Bobryshev and Lord 1995
,1998
; Bobryshev 2000
; Bobryshev and Lord 2002
) and suggested that some DCs interact with T cells directly within atherosclerotic lesions, whereas others may migrate to regional lymph nodes to activate T cells (Bobryshev and Lord 1998
; Bobryshev 2000
).
Recent studies revealed that natural killer T (NKT) cells are involved in atherosclerosis (Nakai et al. 2004; Tupin et al. 2004
). Earlier studies implicated NKT cells in the regulation of autoimmunity, particularly in diabetes and experimental allergic encephalomyelitis in animal models (van Kaer, 2004
). NKT cells comprise a heterogeneous subpopulation of T cells that coexpress a TCR and natural killer (NK) surface antigen CD161 in humans and NK1.1 in mice (van Kaer 2004
). NKT cells are CD1d-restricted and express an evolutionarily conserved TCR with invariant
-chain (V
14-J
18 in mouse and V
24-J
18 in human). NKT cells react to exogenous
-galactosylceramide (
GalCer), which is presented by the monomorphic HLA class-Ilike molecule CD1d (van Kaer, 2004
). The treatment of apoliprotein Edeficient (apoE/) mice with
GalCer resulted in accelerated atherosclerosis (Nakai et al. 2004
; Tupin et al. 2004
) accompanied by the recruitment of NKT cells to aortic atherosclerotic-like lesions in apoE/ mice. Similar treatment of CD1d/ apoE/ mice had no impact on disease progression, suggesting that the effect of
GalCer on atherosclerosis progression is CD1d-dependent (Nakai et al. 2004
; Tupin et al. 2004
).
No previous studies have investigated whether NKT cells are present in human atherosclerotic lesions and, if so, whether there is an association between NKT cells and DCs. In the present study, using immunohistochemical techniques, we examined human carotid artery specimens collected as previously reported (Bobryshev and Lord 1998). For immunohistochemical examination, unfixed specimens were immediately embedded in OCT compound, rapidly frozen in liquid nitrogen, and stored at 70C until cryostat sectioning. For the present study, frozen sections were obtained from eleven carotid artery specimens containing advanced atherosclerotic lesions and adjacent normal-appearing arterial wall. The material was collected in accordance with the Helsinki Declaration of 1975, and some characteristics of these specimens have been reported previously (Bobryshev and Lord 2002
). All atherosclerotic plaques selected for the present analysis were unstable plaques according to the criteria described by Yilmaz et al. (2004)
. The sections were single and double immunostained. The total population of T cells was identified with anti-CD3 (Dako, Glostrup, Denmark; dilution 1:50). NKT cells were identified using anti-NK surface antigen CD161 (Santa Cruz Biotechnology, Santa Cruz, CA; 1:100). DCs were identified with anti-CD1a (Dako; 1:50) and fascin (Dako; 1:100). CD1d expression was visualized using anti-CD1d (Santa Cruz Biotechnology). Macrophages, smooth muscle cells, and endothelial cells were identified as previously described (Bobryshev and Lord 1998
,2002
). For single immunostaining, the avidin-biotin immunoperoxidase method was used as previously detailed (Bobryshev and Lord 1995
). Double immunostaining included combinations of peroxidase-anti-peroxidase and alkaline phosphatase-anti-alkaline phosphatase techniques, which were carried out as described previously (Bobryshev and Lord 1998
,2002
). Negative controls were also carried out as described previously (Bobryshev and Lord 1998
).
The distribution patterns of DCs in atherosclerotic lesions were similar to those described previously (Bobryshev and Lord 1998). Consistent with our previous observations, DCs were located predominantly in plaque shoulders containing inflammatory infiltrates consisting of T cells and macrophages. Comparison of consecutive sections stained with anti-CD1d and with different cell-typespecific antibodies confirmed that CD1d expression is restricted to DCs (Figures 1A and 1B). Immunohistochemical examination demonstrated that there were no NKT cells in the non-diseased arterial sites, but atherosclerotic plaques contained NKT cells (Figures 1C and 1D). Although NKT cells represented a minor cell population among T cells (0.32%), they were found in all specimens studied. NKT cells were observed most often within inflammatory infiltrates in the deep portions of plaques underlying the necrotic cores and in plaque shoulders. In plaque shoulders, the colocalization of CD1d+ DCs with NKT cells was identified (Figure 1E). Electron microscopic examination of areas corresponding to zones where DCs were colocalized with NKT cells demonstrated direct contacts between DCs and lymphocytes, some of which contained well-developed cytoplasm typical of NKT cells (Figures 2A2C). In clusters of DCs with lymphocytes, cisterns of the DC tubulovesicular system were hypertrophied (Figures 2A2C), suggesting that DCs were activated.
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Emerging evidence suggests that DCs are involved in the regulation of the helper T cell Th1/Th2 balance and may interact with effector cells of innate immunity (Creusot and Mitchison 2004). It has been reported that DCs can activate NKT cells by presenting
GalCer in association with CD1d (Creusot and Mitchison, 2004
).
DCs may represent a useful tool for the regulation of NKT cell function in atherosclerosis. DCs pulsed with GalCer are used today for cancer immunotherapy (Nieda et al. 2004
). In one such technique, DCs pulsed with
GalCer ex vivo are returned to the bloodstream (Nieda et al. 2004
). In atherosclerosis, in contrast to cancer immunotherapy, NKT cells need to be suppressed (Nakai et al. 2004
; Tupin et al. 2004
). It is known that the presentation of MHC complexes to T cells by DCs in the absence of costimulatory signals does not lead to T cell activation but, quite the opposite, leads to the deletion or inactivation of these T cells (Austyn 1998
; Lotze and Thomson 2001
). Perhaps, for atherosclerosis immunotherapy, costimulatory molecules on DCs pulsed with
GalCer need to be downregulated/ligated before they are returned to the bloodstream.
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Acknowledgments |
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Footnotes |
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Literature Cited |
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Austyn JM (1998) Dendritic cells. Curr Opin Hematol 5:315[Medline]
Bobryshev YV (2000) Dendritic cells and their involvement in atherosclerosis. Curr Opin Lipidol 11:511517[CrossRef][Medline]
Bobryshev YV, Lord RSA (1995) Ultrastructural recognition of cells with dendritic cell morphology in human aortic intima. Contacting interactions of vascular dendritic cells in athero-resistant and athero-prone areas of the aorta. Arch Histol Cytol 58:307322[Medline]
Bobryshev YV, Lord RSA (1998) Mapping of vascular dendritic cells in atherosclerotic arteries suggests their involvement in local immune-inflammatory reactions. Cardiovasc Res 37:799810[CrossRef][Medline]
Bobryshev YV, Lord RS (2002) Expression of heat shock protein-70 by dendritic cells in the arterial intima and its potential significance in atherogenesis. J Vasc Surg 35:368375[CrossRef][Medline]
Creusot RJ, Mitchison NA (2004) How DCs control cross-regulation between lymphocytes. Trends Immunol 25:126131[CrossRef][Medline]
Hansson GK, Libby P, Schonbeck U, Yan ZQ (2002) Innate and adaptive immunity in the pathogenesis of atherosclerosis. Circ Res 91:281291
Lotze MT, Thomson AW, eds (2001) Dendritic Cells: Biology and Clinical Applications. 2nd ed. San Diego, Academic Press
Millonig G, Niederegger H, Rabl W, Hochleitner BW, Hoefer D, Romani N, Wick G (2001) Network of vascular-associated dendritic cells in intima of healthy young individuals. Arterioscler Thromb Vasc Biol 21:503508
Nakai Y, Iwabuchi K, Fujii S, Ishimori N, Dashtsoodol N, Watano K, Mishima T, et al. (2004) Natural killer T cells accelerate atherogenesis in mice. Blood 104:20512059
Nieda M, Okai M, Tazbirkova A, Lin H, Yamaura A, Ide K, Abraham R, et al. (2004) Therapeutic activation of Valpha24+Vbeta11+ NKT cells in human subjects results in highly coordinated secondary activation of acquired and innate immunity. Blood 103:383389
Porcelli SA (1995) The CD1 family: a third lineage of antigen-presenting molecules. Adv Immunol 59:198[Medline]
Tupin E, Nicoletti A, Elhage R, Rudling M, Ljunggren HG, Hansson GK, Berne GP (2004) CD1d-dependent activation of NKT cells aggravates atherosclerosis. J Exp Med 199:417422
van Kaer L (2004) Natural killer T cells as targets for immunotherapy of autoimmune diseases. Immunol Cell Biol 82:315322[CrossRef][Medline]
Yilmaz A, Lochno M, Traeg F, Cicha I, Reiss C, Stumpf C, Raaz D, et al. (2004) Emergence of dendritic cells in rupture-prone regions of vulnerable carotid plaques. Atherosclerosis 176:101110[CrossRef][Medline]