Inflammatory mechanisms in atherosclerosis

Authors


Goran K. Hansson, Karolinska Institutet, Center for Molecular Medicine L8:03, Department of Medicine, Karolinska University Hospital, SE-17176 Stockholm, Sweden.
Tel.: +46 8 51776222; fax: +46 8 313147.
E-mail: Goran.Hansson@ki.se

Abstract

Summary.  Atherosclerosis, the cause of myocardial infarction, stroke and ischemic gangrene, is an inflammatory disease. When LDL accumulates in the intima, it activates the endothelium to express leukocyte adhesion molecules and chemokines that promote recruitment of monocytes and T cells. Monocyte-derived macrophages upregulate pattern recognition receptors, including scavenger receptors that mediate uptake of modified LDL, and Toll-like receptors, which transmit activating signals leading to release of cytokines, proteases, and vasoactive molecules. T cells in lesions recognize local antigens and mount Th1 responses with secretion of pro-inflammatory cytokines, thus contributing to local inflammation and growth of the plaque. Intensified inflammatory activation may lead to local proteolysis, plaque rupture, and thrombus formation, triggering ischemia and infarction. Inflammatory markers are already used to monitor the disease process and anti-inflammatory therapy may be useful to control disease activity.

Formation of atherosclerotic plaques

When plasma levels of the cholesterol-rich very low density and low-density lipoproteins rise, low-density lipoprotein (LDL) particles are retained in the extracellular matrix of the artery, where they become targets for oxidative and enzymatic attack [1]. Phospholipids released from LDL may activate endothelial cells to express several types of leukocyte adhesion molecules [2]. Once adherent to the endothelium, leukocytes migrate into the underlying intima in response to chemokines including MCP-1, RANTES, and fractalkine [3]. Macrophage-colony stimulating factor (M-CSF) induces monocytes to differentiate into macrophages [4]. This process is associated with upregulation of pattern recognition receptors including scavenger receptors (SR) and Toll-like receptors (TLR) [4]. SR mediate uptake of oxidized LDL particles leading to the formation of foam cells, while TLR initiate signalling cascades that lead to inflammatory activation. This may cause release of vasoactive molecules such as nitric oxide, endothelins, and several eicosanoids including leukotrienes [4]. The latter have been implicated in atherosclerosis based on their production in atheroma, effects on experimental atherosclerosis, and associations between genetic polymorphism in the leukotriene pathway and coronary artery disease.

In the T-cell population of human plaques, CD4+ cells dominate over CD8+ cells [5]. Most T cells are of the TCRαβ type and often found in clusters in shoulder regions of the lesion. MHC class II-expressing macrophages and dendritic cells can be detected adjacent to these T cells, suggesting an ongoing immune activation [5,6]. T cells in atheroma display an activated/memory phenotype [5] and the proportion of activated T cells is particularly high in culprit lesions causing acute coronary syndrome [7,8].

Role of T and B cells in mouse models of disease

Rag-1−/−, Rag-2−/− and scid/scid mice lacking mature T and B cells have been crossed with apoE−/− or LDLR−/− mice to study the role of lymphocytes in atherosclerosis [5]. Compared with immunocompetent apoE−/− mice, the immunodeficient ones develop much smaller lesions. Transfer of CD4+ T cells from older apoE−/− mice to apoE−/−scid/scid mice restored lesion development almost to the levels found in apoE−/− mice, indicating that CD4+ cells are proatherogenic in the context of hypercholesterolemia [9].

CD8+ T cells, although less abundant than CD4+ ones, may also contribute to atherosclerosis. Thus, activation of antigen-specific CD8+ cells accelerates atherosclerosis in mice [10]. CD137-dependent activation of CD8+ T cells leads to infiltration of such cells into plaques [11].

Antigens implicated in atherogenesis

Several antigens have been associated with atherosclerosis. Ten per cent of CD4+ T-cell clones derived from human plaques respond to components of oxidized LDL as an MHC class II-restricted antigen [5,12]. Other plaque T cells react to heat shock protein (HSP)-60/65 that may be endogenous or derived from several pathogens [5].

Antibody titers to oxidized LDL are increased in atherosclerotic patients and experimental animals, with antibodies to many different native and MDA-modified peptide sequences of apolipoprotein B [13,14] as well as oxidized phospholipids [15]. Natural antibodies to phosphocholine are produced by B1 cells, a subset of B cells, even without immunization. They are usually IgM, increase upon immunization, and cross-react between phospholipids on apoptotic cells, oxidized LDL, and the capsule of Streptococcus pneumoniae [16]. All these data show that several T-cell-dependent and -independent antigens are present in atherosclerotic plaques and elicit immune responses that may either be proatherosclerotic or atheroprotective. The choice of pathway depends on the local environment rather than the antigen.

Predominance of Th1 effector cells and cytokines

Data from human and animal studies show a predominant Th1 pattern of cellular immune responses in atherosclerosis. The prototypic Th1 cytokine, IFN-γ is produced locally, as are the Th1 inducing cytokines IL-12 and IL-18 [5]. Th1 differentiation depends on the transcription factor, T-bet. Its absence leads to significant reduction of atherosclerosis [17]. Analogously, deletion of IL-12 and IL-18 or administration of a Th1 inhibitory drug, pentoxifyllin [18] reduces disease.

IFN-γ is a major proatherogenic Th1 cytokine. It promotes macrophage and endothelial activation with production of adhesion molecules, cytokines, chemokines, radicals, proteases, and coagulation factors, and inhibits cell proliferation, collagen production, and cholesterol efflux [19]. Targeted deletion of IFN-γ or its receptor reduces disease, while administration of recombinant IFN-γ accelerates lesion development in hypercholesterolemia and after allotransplantation. Although Th1 cells are major sources of IFN-γ, it can also be produced by CD8+ T cells, NKT cells, NK cells, and IL-18-stimulated smooth muscle cells [20,21].

TNF is produced by Th1 cells, macrophages, and NK cells. It is pro-inflammatory and cytotoxic, and inhibits metabolic enzymes including lipoprotein lipase. Gene targeting of TNF leads to reduced atherosclerosis [21]. The related cytokine lymphotoxin is also produced by Th1 cells and exerts similar effects, as do other members of the TNF superfamily. Ligation of lymphotoxin receptors in the liver by the TNF superfamily member, LIGHT, modulates lipoprotein metabolism by inhibiting hepatic lipase [22]. Blocking TNF-like CD40L reduces atherosclerosis in mice [23].

Regulatory T cells and anti-inflammatory cytokines

Regulatory T cells are implicated in the maintenance of self-tolerance and control of autoimmunity. Recent reports show that regulatory T cells infiltrate lesions of apoE−/− mice [24], that regulatory T cells type 1 (Tr1) may inhibit lesion growth [25], and that TGF-β producing ‘natural’ regulatory T cells inhibit atherosclerosis [26].

TGF-β

TGF-β is a pluripotent cytokine secreted by a number of cells, including regulatory T cells, macrophages, dendritic cells, platelets, endothelial cells, and smooth muscle cells [5,19]. TGF-β is known to be atheroprotective because of its inhibition of T-cell activation [27].

IL-10

IL-10 is produced by several immune cells, inhibits secretion of other cytokines, and is immunosuppressive. It is also atheroprotective (reviews in Refs [5,19]). IL-10 may promote plaque stability: IL-10-deficient mice display increased plaque protease activity, decreased collagen, increased tissue factor activity and enhanced thrombogenicity [28,29].

NKT cells

The NKT cell recognizes lipids displayed on CD1 molecules on the surface of the antigen-presenting cell. Recent data suggest that NKT cells contribute to atherosclerosis [30]. CD1-defective mice develop smaller lesions, whereas activation of NKT cells aggravates lesion formation. Therefore, it is possible that NKT cell recognition of local lipid antigens contributes to atherosclerosis.

B cells and antibodies

Few B cells are detected in atherosclerotic plaques [5] but substantial amounts of B cells and plasma cells are found in periadventitial lymphoid infiltrates surrounding advanced lesions, where they may form tertiary lymphoid structures with germinal centers [31]. Several lines of evidence support the hypothesis that humoral immunity protects against atherosclerosis. Injections of intravenous immunoglobulin preparations inhibit atherosclerosis in apoE−/− mice [32,33]. Splenectomy increases lesion development in apoE−/− mice, whereas B-cell transfer ameliorates atherosclerosis [34]. Similarly, transfer of bone marrow from mice lacking B cells increases lesion growth in LDLR−/− mice [35]. These effects may at least partly depend on B cells producing antibodies to oxidized LDL [36].

Plaque activation elicits thrombosis and clinical symptoms

Acute clinical complications of atherosclerosis such as myocardial infarction and ischemic stroke are caused by the formation of a thrombus on the plaque [8]. Patients with acute coronary syndromes display signs of inflammation, with elevated levels of circulating cytokines, acute phase reactants, and activated T cells [8]. Activated macrophages, T cells, and mast cells are found at sites of plaque rupture and produce several types of molecules that can destabilize lesions: pro-inflammatory cytokines, proteases, coagulation factors, radicals, and vasoactive molecules [37]. Atherosclerotic lesions of mice with increased T-cell activation display reduced amounts of mature, cross-linked collagen and smaller fibrous caps. This was due to inhibition of lysyl oxidase, an enzyme needed for collagen cross-linking, by cytokines of activated T cells [38].

Three types of proteases have been implicated in plaque activation [19,37]. Several types of proteases are found in the plaque, including matrix metalloproteinases, cysteine proteases, and chymase. They degrade its matrix and may contribute to rupture, and thrombosis.

Immunomodulatory strategies to prevent or treat atherosclerosis

Several groups have reported a reduction of atherosclerosis in hypercholesterolemic rabbits and mice after immunization with oxidized LDL [39]. T-cell-dependent IgG antibody production to MDA-LDL is increased after immunization, indicating that antigen-specific CD4+ T cells are activated. Such an effect probably involves immune recognition of protein-derived peptide fragments. Indeed, immunization with peptides from the LDL protein, apoB100, reduced lesion development in apoE−/− mice [38].

Conclusions

Atherosclerosis is an inflammatory disease. Immune cells – macrophages, T cells, NKT cells, dendritic cells and mast cells – infiltrate lesions at all stages. Disease development is inhibited when recruitment of immune cells is inhibited, while it is accelerated by reagents, cytokines and antigens, which activate these cells. Pathogen-associated molecular patterns may activate lesion macrophages, while several antigens including components of LDL induce T-cell activation and antibody formation. Inflammatory effector mechanisms operate largely through cytokine secretion and their activation may conceivably cause plaque rupture, thrombosis, and acute ischemic symptoms. Anti-inflammatory and immunosuppressive mechanisms inhibit atherosclerosis and may be attractive targets for disease prevention and/or treatment. They include anti-inflammatory cytokines, protective antibodies, and regulatory T cells, and may be induced by immunization. Interestingly, several drugs currently used to treat atherosclerotic patients operate at least partly through immune inhibitory mechanisms.

Acknowledgments

Our work is supported by the Swedish Research Council, Heart-Lung Foundation, the European Union, and the Leducq Foundation.

Disclosure of Conflict of Interests

The author states that he has no conflict of interest.

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