Interleukin-10 and coronary disease

Understanding of the pathophysiology of atherosclerosis has changed markedly over the past few decades. It is now widely accepted that inflammation plays a fundamental role in the genesis and development of atherosclerosis. Inflammatory mechanisms also appear to determine clinical presentation and disease outcome. Atherosclerotic lesions have high concentrations of inflammatory cells (T lymphocytes and activated macrophages) as well as an abundance of pro-inflammatory cytokines [interleukin (IL)-1, IL-6, IL-8, interferon-gamma, tumor necrosis factor-alpha, etc.] that modulate local inflammatory responses. These may also alter plaque stability and facilitate the development of acute cardiovascular events. The role of anti-inflammatory cytokines in this context remains to be studied. IL-10 is an anti-inflammatory cytokine synthesised by T-lymphocytes and macrophages and has other anti-inflammatory effects. IL-10 expression within human atherosclerotic plaques has been demonstrated and animal experiments have shown that low levels of IL-10 lead to the development of extensive and unstable atherosclerotic lesions. Currently available evidence suggests a potential protective role for IL-10 in atherosclerosis. This new perspective on coronary disease as a chronic inflammatory process may open new avenues for the management of ischemic heart disease.     

Interaction between pro-inflammatory and anti-inflammatory cytokines in insulin-producing cells

Pro-inflammatory cytokines cause beta-cell dysfunction and death. The aim of this study was to investigate the interactions between different pro- and anti-inflammatory cytokines and their effects on apoptotic beta-cell death pathways. Insulin-producing RINm5F cells were exposed to different combinations of cytokines. Gene expression analyses of manganese superoxide dismutase (MnSOD) and inducible nitric oxide synthase (iNOS) were performed by real-time RT-PCR. Cell viability was measured by the MTT assay, NFkappaB activation using a SEAP reporter gene assay, protein expression by western blotting and caspase-3 activity using the DEVD cleavage method. IL-1beta, tumour necrosis factor alpha (TNFalpha) and a combination of all three pro-inflammatory cytokines increased while IFNgamma alone did not affect NFkappaB activity and iNOS gene and protein expression. Interestingly, the anti-inflammatory cytokines IL-4, IL-13 and IL-10 decreased IL-1beta-stimulated NFkappaB activation and iNOS expression. IL-1beta, TNFalpha and the pro-inflammatory cytokine combination also increased MnSOD gene and protein expression. But IL-4, IL-13 and IL-10 did not affect MnSOD expression and did not modulate IL-1beta-stimulated MnSOD expression. Caspase-3 activity was increased by IL-1beta and the pro-inflammatory cytokine combination, and to a lesser extent by TNFalpha. In contrast, IFNgamma had no effect on caspase-3 activity. IL-4, IL-13 and IL-10 decreased caspase-3 activity and increased viability of insulin-producing cells treated with pro-inflammatory cytokines. The anti-inflammatory cytokines counteracted the cytotoxic effects of pro-inflammatory cytokines in insulin-producing cells. This was achieved through the reduction of nitrosative stress. Thus, a balance between the anti-inflammatory and the pro-inflammatory cytokines is of crucial importance for the prevention of pancreatic beta-cell destruction.     

Inducible nitric oxide synthase colocalizes with signs of lipid oxidation/peroxidation in human atherosclerotic plaques

OBJECTIVE: Advanced human atherosclerotic plaques are characterized by the abundant presence of the autofluorescent non-soluble lipid pigment ceroid, consisting of oxidized lipoproteins. The aim of the present study was to examine the topographical and cellular distribution of inducible nitric oxide synthase (iNOS or NOS II) within different stages of atherosclerosis and its colocalization with ceroid deposits and nitrotyrosine. METHODS AND RESULTS: Different stages of atherosclerosis were studied by immunohistochemistry on whole-mount longitudinal sections of carotid endarterectomy specimens. In the adaptive intimal thickening the predominant cell type were smooth muscle cells. The fatty streaks contained both smooth muscle cells and macrophages with an extremely low NOS II immunoreactivity. The advanced atherosclerotic plaques however, showed a very dense infiltration by macrophages, of which a subpopulation expressed NOS II as a vesicular immunoreactivity in their cytoplasm. These were mainly present around the necrotic core, in association with ceroid accumulation and nitrotyrosine. Fluorescence quenching microscopy showed the presence of NOS II on autofluorescent ceroid vesicles in the macrophages. Large extracellular ceroid granules were not NOS II immunoreactive. NOS II mRNA was detected by RT-PCR and the protein by Western blot in the plaque tissue but not in mammary arteries used as controls. CONCLUSION: Ceroid, nitrotyrosine and NOS II colocalized in late stages of atherosclerosis and were found around the necrotic core in the plaque. This could suggest that NOS II expression in macrophages is involved in oxidation and peroxidation of lipids, leading to ceroid formation.     

A functional role for inducible costimulator (ICOS) in atherosclerosis

BACKGROUND: Lymphocytes appear to influence atherosclerosis by altering cytokine production. Whereas primary lymphocyte activation requires T cell receptor ligation, costimulatory signals also appear requisite for generation of a functional T cell response. Inducible costimulator (ICOS) is a newly discovered T cell molecule with a dual role in immune mediated disorders. Herein, we tested the importance of ICOS in atherosclerosis. METHODS AND RESULTS: Atherosclerotic plaques from ApoE-KO mice were studied immunohistochemically for the presence and localization of ICOS and its receptors and its expression in splenocytes. ApoE-KO mice were immunized with human ICOS/Fc-chimera or non-fused Fc and either provided a chow diet for 6 weeks, or a high fat diet for 8 weeks. ICOS and its ligand were abundantly expressed within plaques from ApoE-KO mice: Spleen cells from atherosclerotic mice exhibited lowered constitutive expression of ICOS yet priming with oxLDL enhanced ICOS expression dose-dependently. In mice induced to develop fatty streaks and to generate ICOS blocking antibodies, early atherosclerosis was increased by approximately 77% whereas upon inducing more advanced lesions, the increase in plaque area upon ICOS blockade group was approximately 36%. IFN-gamma secretion by oxLDL-primed splenocytes in ICOS-immunized mice increased whereas IL-10 secretion diminished as compared to control animals. A similar trend in cytokine production was evident in the lesion by immunohistochemistry. CONCLUSION: ICOS appears as an influential costimulatory pathway in atherosclerosis that may play a protective rather that a proatherogenic role.     

Toll-like receptor 7 stimulation by imiquimod induces macrophage autophagy and inflammation in atherosclerotic plaques

Atherosclerotic plaques tend to rupture as a consequence of a weakened fibrous cap, particularly in the shoulder regions where most macrophages reside. Macrophages express Toll-like receptors to recognize pathogens and eliminate intracellular pathogens by inducing autophagy. Because Toll-like receptor 7 (TLR7) is thought to be expressed in macrophages but not in smooth muscle cells (SMCs), we investigated whether induction of macrophage autophagic death by TLR7 ligand imiquimod can affect the composition of atherosclerotic plaques in favor of their stability. Immunohistochemical staining of human carotid plaques as well as Western blotting of cultured macrophages and SMCs confirmed that TLR7 was expressed in macrophages, but not in SMCs. In vitro experiments showed that only TLR7 expressing cells underwent imiquimod-induced cell death, which was characterized by autophagosome formation. Imiquimod-treated macrophages activated nuclear factor-κB (NF-κB) and released pro-inflammatory cytokines and chemokines. This effect was inhibited by the glucocorticoid dexamethasone. Imiquimod-induced cytokine release was significantly decreased in autophagy-deficient macrophages because these cells died by necrosis at an accelerated pace. Local in vivo administration of imiquimod to established atherosclerotic lesions in rabbit carotid arteries induced macrophage autophagy without induction of cell death, and triggered cytokine production, upregulation of vascular adhesion molecule-1, infiltration of T-lymphocytes, accumulation of macrophages and enlargement of plaque area. Treatment with dexamethasone suppressed these pro-inflammatory effects in vivo. SMCs and endothelial cells in imiquimod-treated plaques were not affected. In conclusion, imiquimod induces macrophage autophagy in atherosclerotic plaques, but stimulates plaque progression through cytokine release and enhanced infiltration of inflammatory cells.     

Role of ERK/MAPK signalling pathway in anti-inflammatory effects of Ecklonia cava in activated human mast cell line-1 cells

Objective:The anti-inflammatory effects of Ecklonia cava(EC)and its mechanism of action were examined in phorbol-12 myristate 13-acetate(30 nmol/L)and A23187(1μmol/L)(PMACI)stimulated human mast cell line-1 cells.Methods:Nitric oxide content,inducible nitric oxide synthase and cyclooxygenase-2 protein expression,pro-inflammatory cytokines including IL-1β,TNF-α,and IL-6 mRNA and protein expressions were determined.In addition,extracellular regulated protein kinases/mitogen-activated protein kinase(ERK/MAPK)activation was examined.Results:EC dose-dependently suppressed inducible nitric oxide synthase and cyclooxygenase-2 protein expression and subsequently it reduces nitric oxide content in PMACI stimulated human mast cell line-1 cells.EC dose-dependently inhibited the mRNA as well as protein expression of TNF-α,IL—1β,and TL-6 in the PMACI stimulated human mast cell line-1 cells without any cytotoxic effect.Furthermore,EC significantly inhibited PMACI induced phosphorylation of ERK1/2 in a dose-dependent manner without affecting the total protein levels.Conclusions:EC exert its anti-inflammatory actions via inhibition of ERK/MAPK signalling pathway,suggesting that EC is a potent and efficacious anti-inflammatory agent for mast cellmediated inflammatory diseases.     

通心络对实验性家兔主动脉粥样斑块内血管内皮生长因子表达的影响

通过构建实验性家兔主动脉粥样硬化模型，观察通心络及辛伐他汀对血清氧化型低密度脂蛋白、一氧化氮、一氧化氮合酶和乳酸脱氢酶及血管形态学的影响，并采用免疫组织化学、逆转录一聚合酶链反应及Western Blot蛋白印迹等方法，从不同水平观察通心络及辛伐他汀对动脉粥样癍块血管内皮生长因子表达的影响。结果发现，通心络减少动脉粥样斑块血管内皮生长因子表达，降低血清氧化型低密度脂蛋白及乳酸脱氢酶浓度，提示通心络可增加粥样斑块稳定性，延缓动脉粥样硬化的进程。     

TWEAK can induce pro-inflammatory cytokines and matrix metalloproteinase-9 in macrophages.

The expression of TWEAK (TNFSF12) and TweakR/Fn14 was detected in regions rich in macrophage/foam cells in atherosclerotic plaques. The role of TWEAK in monocytes in relation to atherogenesis was investigated by analyzing the cellular events induced by TWEAK in a human macrophage-like cell line, THP-1. TWEAK induced various molecular mediators of atherogenesis, such as IL-6, MCP-1, IL-8 and MMP-9, and the induction was augmented by interferon-gamma. TWEAK-induced activation of MMP-9 was mediated by activation of NF-kappaB. These results suggest that TWEAK is involved in atherosclerosis by inducing pro-inflammatory cytokines and extracellular matrix degrading enzymes, which reduce plaque stability.     

白细胞介素18和基质金属蛋白酶9在人冠状动脉粥样硬化斑块中的表达

目的 探讨白细胞介素18和基质金属蛋白酶9在不同程度的人冠状动脉病变中的表达情况及意义.方法收集14例尸检的人冠状动脉共48个冠状动脉标本,根据HE染色将标本分为不稳定斑块组、稳定斑块组和正常对照组.用免疫组织化学及逆转录聚合酶链反应检测白细胞介素18和基质金属蛋白酶9蛋白及mRNA在以上不同类型标本中的分布表达.结果 白细胞介素18和基质金属蛋白酶9表达于冠状动脉粥样硬化斑块区,在不稳定斑块中的表达明显高于稳定斑块.并且二者的表达强度呈正相关.结论 白细胞介素18和基质金属蛋白酶9可能参与冠状动脉粥样硬化斑块的发生、发展,与斑块不稳定性密切相关.并且二者协同参与粥样斑块发展.     

Nitric oxide mediates cytokine-induced inhibition of insulin secretion by human islets of Langerhans.

Cytokines have been implicated as immunological effector molecules that mediate beta cell destruction associated with insulin-dependent diabetes mellitus. In this report we demonstrate that the cytokine combination of human recombinant interleukin 1 beta (IL-1 beta), tumor necrosis factor alpha (TNF-alpha), and interferon gamma (IFN-gamma) induces the formation of nitric oxide by human islets. This combination of cytokines stimulates both the formation of the nitric oxide derivative, nitrite, and the accumulation of cGMP by human islets. The nitric oxide synthase inhibitor NG-monomethyl-L-arginine prevents formation of both cGMP and nitrite. IL-1 beta and IFN-gamma are sufficient to induce nitric oxide formation by human islets, whereas TNF-alpha potentiates nitrite production. This combination of cytokines (IL-1 beta, TNF-alpha, and IFN-gamma) also influences insulin secretion by human islets. Pretreatment of human islets with low concentrations of this cytokine combination (IL-1 beta at 15 units/ml, 0.7 nM TNF-alpha, and IFN-gamma at 150 units/ml) appears to slightly stimulate insulin secretion. Higher concentrations (IL-1 beta at 75 units/ml, 3.5 nM TNF-alpha, and IFN-gamma at 750 units/ml) inhibit insulin secretion from human islets, and the inhibitory effect is prevented by NG-monomethyl-L-arginine. This higher concentration of cytokines also induces the formation of an electron paramagnetic resonance-detectable g = 2.04 axial feature by human islets that is characteristic of the formation of an iron-dithio-dinitrosyl complex. The formation of this complex is prevented by NG-monomethyl-L-arginine, thus confirming that this cytokine combination induces the formation of nitric oxide by human islets. These results indicate that nitric oxide mediates the inhibitory effects of cytokines on glucose-stimulated insulin secretion by human islets and suggest that nitric oxide may participate in beta-cell dysfunction associated with insulin-dependent diabetes mellitus.     

Expression of IL-17A in human atherosclerotic lesions is associated with increased inflammation and plaque vulnerability

A chronic (auto)immune response is the critical mechanism in atherosclerosis. Interleukin-17A is a pivotal effector cytokine, which modulates immune cell trafficking and initiates inflammation in (auto)immune and infectious diseases. However, expression of IL-17A in the context of human atherosclerosis has hardly been explored. Carotid artery plaques were collected from 79 patients undergoing endarterectomy. Patients were grouped according to their symptomatic status (TIA, stroke), plaque morphology and medication. Quantitative RT-PCR was used to analyze tissue inflammation and immunohistochemistry to assess cellular source of IL-17A expression and lesion morphology. Carotid plaques from patients with ischemic symptoms were characterized by a highly activated inflammatory milieu including accumulation of T cells (p = 0.04) and expression of IL-6 and VCAM1 (p = 0.02, 0.01). Expression of IL-17A and its positive regulators IL-21 and IL-23 was present in atherosclerotic lesions, significantly upregulated in atheromas of symptomatic patients (p = 0.005, 0.004, 0.03), and expression of IL-17A and IL-21 showed a strong correlation (p = 0.002, r = 0.52). The cellular sources of lesional IL-17A expression are T cells, macrophages, B cells and plasma cells. Vulnerable/ruptured (complicated) plaques were significantly associated with IL-17A expression levels (p = 0.003). In addition, IL-17A showed a marked negative correlation with the potent anti-inflammatory/atheroprotective cytokine IL-10 (p = 0.0006, r = −0.46). Furthermore, treatment with a HMG-CoA reductase inhibitor or acetylsalicylic acid showed reduced levels of IL-21, IL-23 and VCAM1 (all p < 0.05), but did not influence IL-17A. The association of IL-17A with ischemic symptoms and vulnerable plaque characteristics suggests that the pro-inflammatory cytokine IL-17A may contribute to atherosclerosis und plaque instability.     

Interleukin-18/interleukin-18 binding protein signaling modulates atherosclerotic lesion development and stability

Interleukin (IL)-18 is the interferon-gamma-inducing factor and has other proinflammatory properties. The precise role of IL-18 in immunoinflammatory diseases remains poorly understood. In this study, we show that in vivo electrotransfer of an expression-plasmid DNA encoding for murine IL-18 binding protein (BP) (the endogenous inhibitor of IL-18) prevents fatty streak development in the thoracic aorta of apoE knockout mice and slows progression of advanced atherosclerotic plaques in the aortic sinus. More importantly, transfection with the IL-18BP plasmid induces profound changes in plaque composition (decrease in macrophage, T cell, cell death, and lipid content and increase in smooth muscle cell and collagen content) leading to a stable plaque phenotype. These results identify for the first time a critical role for IL-18/IL-18BP regulation in atherosclerosis and suggest a potential role for IL-18 inhibitors in reduction of plaque development/progression and promotion of plaque stability. The full text of this article is available at http://www.circresaha.org.     

Cytokine profiles and T cell function in acute coronary syndromes

AIMS: In advanced human atherosclerotic plaques infiltrating T cells congregate at sites of plaque rupture. However, little is known about the systemic activation of circulating T cells in acute coronary syndromes as a prerequisite for recruitment to atherosclerotic lesions. METHODS AND RESULTS: As a measure for specific lymphocyte activation we analyzed IFN-gamma production of T cells after stimulation with a superantigen and expression of CXCR-3 and CCR-3 in patients with acute myocardial infarction (AMI), unstable angina (uAP) or stable angina (sAP). Furthermore, concentrations of the circulating cytokines interleukin (IL)-1, IL-6, IL-1beta, IL-12 p70 and RANTES that modify T cell function were measured. In uAP an increased Th1 and a decreased Th2 response was identified by enhanced interferon-gamma generation of T lymphocytes, increased levels of IL-1beta, IL-12 p70 and RANTES and decreased expression of CCR3. In AMI a systemic inflammatory reaction predominates with enhanced expression of the early activation marker CD69 on T lymphocytes and elevated levels of IL-6 and IL-10 that suppress Th1 activation. CONCLUSION: Interferon-gamma production of activated T cells in acute coronary syndromes may, therefore, be governed by the release of specific pro- and anti-lymphocyte activating cytokines.     

A novel chemokine, Leukotactin-1, induces chemotaxis, pro-atherogenic cytokines, and tissue factor expression in atherosclerosis

Chemokines such as monocyte chemoattractant protein (MCP) -1 and interleukin (IL)-8 are known to be involved in various processes in atherosclerosis such as plaque formation, plaque rupture, and thrombus formation. We investigated whether a new chemokine, Leukotactin (LKN)-1, is involved in atherosclerosis. We tested the expression of LKN-1 by immunohistochemical methods in carotid atherosclerotic plaque specimen. Induction of pro-inflammatory cytokines, transmigration, and tissue factor (TF) expression were tested in THP-1 cells and human peripheral blood monocytes treated with recombinant human LKN-1. Immunohistochemical analyses revealed that expression of LKN-1 occurs in regions of plaques rich in foam cells. In a Boyden chamber assay, THP-1 cells treated with 0.01--10 nM of LKN-1 transmigrated through gelatin coated filters in a dose dependent manner. LKN-1 also induced the transient expression of TNF-alpha, IL-8, and MCP-1 within 15 min of the treatment of the THP-1 cells. When peripheral blood monocytes were treated with LKN-1, expression levels of TF and TF-mediated procoagulating activity were induced in a time- and dose-dependent manner. These results raise the possibility that LKN-1 is another chemokine that is involved in the atherogenesis. LKN-1 may chemoattract immune cells into the plaque, induce pro-inflammatory cytokines, and produce thrombi by inducing TF expression.     

Molecular imaging of atherosclerotic plaques using a human antibody against the extra-domain B of fibronectin

Current imaging modalities of human atherosclerosis, such as angiography, ultrasound, and computed tomography, visualize plaque morphology. However, methods that provide insight into plaque biology using molecular tools are still insufficient. The extra-domain B (ED-B) is inserted into the fibronectin molecule by alternative splicing during angiogenesis and tissue remodeling but is virtually undetectable in normal adult tissues. Angiogenesis and tissue repair are also hallmarks of advanced plaques. For imaging atherosclerotic plaques, the human antibody L19 (specific against ED-B) and a negative control antibody were labeled with radioiodine or infrared fluorophores and injected intravenously into atherosclerotic apolipoprotein E-null (ApoE-/-) or normal wild-type mice. Aortas isolated 4 hours, 24 hours, and 3 days after injection exhibited a selective and stable uptake of L19 when using radiographic or fluorescent imaging. L19 binding was confined to the plaques as assessed by fat staining. Comparisons between fat staining and autoradiographies 24 hours after 125I-labeled L19 revealed a significant correlation (r=0.89; P<0.0001). Minimal antibody uptake was observed in normal vessels from wild-type mice receiving the L19 antibody and in atherosclerotic vessels from ApoE-/- mice receiving the negative control antibody. Immunohistochemical studies revealed increased expression of ED-B not only in murine but also in human plaques, in which it was found predominantly around vasa vasorum and plaque matrix. In summary, we demonstrate selective targeting of atheromas in mice using the human antibody to the ED-B domain of fibronectin. Thus, our findings may set the stage for antibody-based molecular imaging of atherosclerotic plaques in the intact organism.     

Oxidised LDL modulates immune-activation by an IL-12 dependent mechanism

Oxidised low density lipoprotein (oxLDL) is one factor that may cause the immune reaction in the artery wall characteristic of atherosclerosis. OxLDL can promote immune activation as determined by enhanced secretion of IFN-gamma and TNF by immune competent cells. We previously demonstrated that Platelet-activating factor (PAF)-like lipids and/or lysophosphatidylcholine (LPC) in OxLDL contribute significantly to this immune activation, but these factors may also inhibit immune activation, at higher concentrations. We here demonstrate that IL-12 induces enhanced IFN-gamma secretion in peripheral blood mononuclear cells (PBMC), with no addition of a specific antigen, as determined by ELISPOT. Antibodies to IL-12 and to MHC class II inhibited both IL-12- and oxLDL-induced IFN-gamma secretion. OxLDL induced IL-12 production in PBMC. In the presence of IL-10, a T helper 2 cytokine, oxLDL induced a decreased IFN-gamma secretion, indicating that the local cytokine-milieu may determine the immunological properties of oxLDL. IL-10 could also be induced by OxLDL. Mononuclear leukocytes were prepared directly from human atherosclerotic plaques obtained at carotid operations. OxLDL had the capacity to induce IL-12, IL-10 and TNF from plaque cells using ELISPOT. Taken together, our data indicate that oxLDL can modulate immune reactivity in atherosclerosis by a nonspecific mechanism. OxLDL can be inhibitory, especially at higher concentrations. However, oxLDL can also promote immune activation by functioning as an adjuvant, potentiating and/or modulating immune-reactions via IL-12 and other cytokines including IL-10. This suggests that a specific T cell epitope in oxLDL is not necessary for oxLDL-induced T cell activation.     

Role of IL-37 in Cardiovascular Disease Inflammation

Inflammation is closely related to the pathogenesis and prognosis of cardiovascular disease (CVD). Interleukin-37 (IL-37), an anti-inflammatory IL-1 family cytokine, shifts cytokine expression from pro- to anti-inflammation via regulation of macrophage polarization and lipid metabolism. In macrophages, IL-37 functions through both intracellular and extracellular pathways to regulate the activity of NF-kB and PTEN as well as the expression of cytokines, including IL-1β, IL-6, and IL-10. Moreover, IL-37 levels are increased in the serum of patients with heart failure, atherosclerosis, and acute coronary syndrome with no evidence of anti-inflammatory effects. However, transgenic overexpression of IL-37 improves cardiac infarct and attenuates atherosclerosis plaque expansion. Hence, it is worthwhile to investigate the precise mechanism and role of IL-37 in the pathogenesis of CVD, which may provide deeper understanding of the inflammatory response in this context. This review summarizes the regulatory role of IL-37 in systematic inflammation induced by CVD and highlights recent advancements in the clinical application of IL-37 as a therapeutic agent or biomarker for diagnosis of CVD.