RESUMEN
The complement anaphylatoxin C5a functions through its two receptors, C5aR (CD88) and C5a receptor-like 2 (C5L2). Their role in atherosclerosis is incompletely understood. We, therefore, analyzed C5aR and probed the yet unknown expression and function of C5L2 in human atherogenesis. Human atherosclerotic plaques obtained by endarterectomy were staged and analyzed for C5L2 and C5aR by IHC and quantitative real-time PCR. C5L2-expressing cells in plaques were mostly macrophages, less neutrophils and endothelial cells, as determined by double immunostaining. Although early influx of C5aR(+) cells was detected, C5L2 levels increased with lesion complexity and colocalized with C5aR and oxidized low-density lipoprotein. Gene expression of C5L2 and C5aR showed similar trends, such as the receptor-expressing cells. The expression of C5L2 in advanced lesions correlated with increased levels of IL-1ß and tumor necrosis factor-α in plaques. Furthermore, in vitro experiments in macrophages from wild-type and C5l2- and C5ar-deficient mice corroborated the contributing role of C5l2 in oxidized low-density lipoprotein-pretreated C5a-induced cytokine expression, as measured by enzyme-linked immunosorbent assay. Finally, C5l2- and C5ar-deficient peripheral blood mononuclear cells showed less arrest on tumor necrosis factor-α-stimulated mouse endothelial cells in vitro when compared with wild-type controls. Taken together, prominent C5L2 expression in advanced atherosclerotic stages directly correlates with high levels of proinflammatory cytokines. This might indicate a proinflammatory role of C5L2 in atherosclerosis that needs to be pursued in the future by applying in vivo mouse models.
Asunto(s)
Citocinas/metabolismo , Placa Aterosclerótica/metabolismo , Receptores de Quimiocina/metabolismo , Animales , Arterias Carótidas/patología , Humanos , Interleucina-1beta/metabolismo , Lipoproteínas LDL/metabolismo , Macrófagos/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Receptor de Anafilatoxina C5a/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
RATIONALE: Neutrophils have been reported to contribute to early atherosclerotic lesion formation. Mechanisms of neutrophil-driven atherosclerosis remain unclear so far. OBJECTIVE: Investigation of the role of the neutrophil granule protein cathelicidin (CRAMP in mouse, LL37 in human) in atherosclerosis. METHODS AND RESULTS: Compared to Apoe(-/-) mice, Cramp(-/-) Apoe(-/-) mice exhibit reduced lesion sizes with lower macrophage numbers. In atherosclerotic aortas, we could detect CRAMP specifically in neutrophils, but not in monocytes or macrophages. By use of intravital microscopy, CRAMP was found to be deposited by activated neutrophils on inflamed endothelium of large arteries. In this location cathelicidins promote adhesion of classical monocytes and neutrophils, but not nonclassical monocytes in a formyl-peptide receptor-dependent manner. CONCLUSIONS: Cathelicidins promote atherosclerosis by enhancement of the recruitment of inflammatory monocytes.
Asunto(s)
Enfermedades de la Aorta/prevención & control , Aterosclerosis/prevención & control , Catelicidinas/deficiencia , Inflamación/prevención & control , Neutrófilos/metabolismo , Animales , Péptidos Catiónicos Antimicrobianos , Aorta/inmunología , Aorta/metabolismo , Aorta/patología , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/inmunología , Enfermedades de la Aorta/metabolismo , Enfermedades de la Aorta/patología , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Aterosclerosis/genética , Aterosclerosis/inmunología , Aterosclerosis/metabolismo , Aterosclerosis/patología , Catelicidinas/genética , Adhesión Celular , Células Cultivadas , Quimiotaxis de Leucocito , Técnicas de Cocultivo , Modelos Animales de Enfermedad , Endotelio Vascular/inmunología , Endotelio Vascular/metabolismo , Humanos , Inflamación/genética , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Fluorescente , Monocitos/inmunología , Monocitos/metabolismo , Neutrófilos/inmunología , Factores de TiempoRESUMEN
Chronic hepatitis leads to liver fibrosis and cirrhosis. Cirrhosis is a major cause of worldwide morbidity and mortality. Macrophages play a key role in fibrosis progression and reversal. However, the signals that determine fibrogenic vs fibrolytic macrophage function remain ill defined. We studied the role of interleukin-4 receptor α (IL-4Rα), a potential central switch of macrophage polarization, in liver fibrosis progression and reversal. We demonstrate that inflammatory monocyte infiltration and liver fibrogenesis were suppressed in general IL-4Rα-/- as well as in macrophage-specific IL-4Rα-/- (IL-4RαΔLysM) mice. However, with deletion of IL-4RαΔLysM spontaneous fibrosis reversal was retarded. Results were replicated by pharmacological intervention using IL-4Rα-specific antisense oligonucleotides. Retarded resolution was linked to the loss of M2-type resident macrophages, which secreted MMP-12 through IL-4 and IL-13-mediated phospho-STAT6 activation. We conclude that IL-4Rα signaling regulates macrophage functional polarization in a context-dependent manner. Pharmacological targeting of macrophage polarization therefore requires disease stage-specific treatment strategies. RESEARCH IN CONTEXT: Alternative (M2-type) macrophage activation through IL-4Rα promotes liver inflammation and fibrosis progression but speeds up fibrosis reversal. This demonstrates context dependent, opposing roles of M2-type macrophages. During reversal IL-4Rα induces fibrolytic MMPs, especially MMP-12, through STAT6. Liver-specific antisense oligonucleotides efficiently block IL-4Rα expression and attenuate fibrosis progression.
Asunto(s)
Subunidad alfa del Receptor de Interleucina-4/metabolismo , Cirrosis Hepática/metabolismo , Cirrosis Hepática/patología , Macrófagos/metabolismo , Transducción de Señal , Animales , Citocinas/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Expresión Génica , Subunidad alfa del Receptor de Interleucina-4/genética , Cirrosis Hepática/tratamiento farmacológico , Cirrosis Hepática/etiología , Activación de Macrófagos/inmunología , Macrófagos/inmunología , Metaloproteinasas de la Matriz/genética , Metaloproteinasas de la Matriz/metabolismo , Ratones , Ratones Noqueados , Células Mieloides/inmunología , Células Mieloides/metabolismo , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/patología , Células RAW 264.7 , Factor de Transcripción STAT6/metabolismo , Bazo/inmunología , Bazo/metabolismo , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismoRESUMEN
We used a novel approach of cytostatically induced leucocyte depletion and subsequent reconstitution with leucocytes deprived of classical (inflammatory/Gr1(hi) ) or non-classical (resident/Gr1(lo) ) monocytes to dissect their differential role in atheroprogression under high-fat diet (HFD). Apolipoprotein E-deficient (Apoe(-/-) ) mice lacking classical but not non-classical monocytes displayed reduced lesion size and macrophage and apoptotic cell content. Conversely, HFD induced a selective expansion of classical monocytes in blood and bone marrow. Increased CXCL1 levels accompanied by higher expression of its receptor CXCR2 on classical monocytes and inhibition of monocytosis by CXCL1-neutralization indicated a preferential role for the CXCL1/CXCR2 axis in mobilizing classical monocytes during hypercholesterolemia. Studies correlating circulating and lesional classical monocytes in gene-deficient Apoe(-/-) mice, adoptive transfer of gene-deficient cells and pharmacological modulation during intravital microscopy of the carotid artery revealed a crucial function of CCR1 and CCR5 but not CCR2 or CX3 CR1 in classical monocyte recruitment to atherosclerotic vessels. Collectively, these data establish the impact of classical monocytes on atheroprogression, identify a sequential role of CXCL1 in their mobilization and CCR1/CCR5 in their recruitment.
Asunto(s)
Enfermedades de la Aorta/inmunología , Aterosclerosis/inmunología , Enfermedades de las Arterias Carótidas/inmunología , Quimiotaxis de Leucocito , Monocitos/inmunología , Receptores de Quimiocina/metabolismo , Traslado Adoptivo , Animales , Aorta/inmunología , Aorta/metabolismo , Aorta/patología , Enfermedades de la Aorta/etiología , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/metabolismo , Enfermedades de la Aorta/patología , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Apoptosis , Aterosclerosis/etiología , Aterosclerosis/genética , Aterosclerosis/metabolismo , Aterosclerosis/patología , Arterias Carótidas/inmunología , Arterias Carótidas/metabolismo , Arterias Carótidas/patología , Enfermedades de las Arterias Carótidas/etiología , Enfermedades de las Arterias Carótidas/genética , Enfermedades de las Arterias Carótidas/metabolismo , Enfermedades de las Arterias Carótidas/patología , Quimiocina CXCL1/metabolismo , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Recuento de Leucocitos , Procedimientos de Reducción del Leucocitos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Monocitos/clasificación , Monocitos/trasplante , Receptores CCR1/metabolismo , Receptores CCR5/metabolismo , Receptores de Quimiocina/deficiencia , Receptores de Quimiocina/genética , Receptores de Interleucina-8B/metabolismo , Transducción de Señal , Factores de TiempoRESUMEN
INTRODUCTION: Treatment of acute lung injury (ALI) remains an unsolved problem in intensive care medicine. As simvastatin exerts protective effects in inflammatory diseases we explored its effects on development of ALI and due to the importance of neutrophils in ALI also on neutrophil effector functions. METHODS: C57Bl/6 mice were exposed to aerosolized LPS (500 µg/ml) for 30 min. The count of alveolar, interstitial, and intravasal neutrophils were assessed 4 h later by flow cytometry. Lung permeability changes were assessed by FITC-dextran clearance and albumin content in the BAL fluid. In vitro, we analyzed the effect of simvastatin on neutrophil adhesion, degranulation, apoptosis, and formation of reactive oxygen species. To monitor effects of simvastatin on bacterial clearance we performed phagocytosis and bacterial killing studies in vitro as well as sepsis experiments in mice. RESULTS: Simvastatin treatment before and after onset of ALI reduces neutrophil influx into the lung as well as lung permeability indicating the protective role of simvastatin in ALI. Moreover, simvastatin reduces the formation of ROS species and adhesion of neutrophils without affecting apoptosis, bacterial phagocytosis and bacterial clearance. CONCLUSION: Simvastatin reduces recruitment and activation of neutrophils hereby protecting from LPS-induced ALI. Our results imply a potential role for statins in the management of ALI.
Asunto(s)
Lesión Pulmonar Aguda/inducido químicamente , Lesión Pulmonar Aguda/tratamiento farmacológico , Endotoxinas/toxicidad , Pulmón/metabolismo , Neutrófilos/inmunología , Simvastatina/farmacología , Lesión Pulmonar Aguda/inmunología , Aerosoles/toxicidad , Albúminas/análisis , Animales , Apoptosis/efectos de los fármacos , Líquido del Lavado Bronquioalveolar/química , Adhesión Celular/efectos de los fármacos , Citometría de Flujo , Fluoresceína-5-Isotiocianato , Lipopolisacáridos , Ratones , Ratones Endogámicos C57BL , Permeabilidad , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Percutaneous transluminal angioplasty with stent implantation is used to dilate arteries narrowed by atherosclerotic plaques and to revascularize coronary arteries occluded by atherothrombosis in myocardial infarction. Commonly applied drug-eluting stents release antiproliferative or anti-inflammatory agents to reduce the incidence of in-stent stenosis. However, these stents may still lead to in-stent stenosis; they also show increased rates of late stent thrombosis, an obstacle to optimal revascularization possibly related to endothelial recovery. Here, we examined the contribution of neutrophils and neutrophilic granule proteins to arterial healing after injury. We found that neutrophil-borne cathelicidin (mouse CRAMP, human LL-37) promoted reendothelization and thereby limited neointima formation after stent implantation. We then translated these findings to an animal model using a neutrophil-instructing, biofunctionalized, miniaturized Nitinol stent coated with LL-37. This stent reduced in-stent stenosis in a mouse model of atherosclerosis, suggesting that LL-37 may promote vascular healing after interventional therapy.