RESUMEN
The rhodamine-based probe R19-S has been shown to react with hypochlorous acid (HOCl) to yield fluorescent R19, but not with some other oxidants including hydrogen peroxide. Here, we further examined the specificity of R19-S and used it for real-time monitoring of HOCl production in neutrophil phagosomes. We show that it also reacts rapidly with hypobromous acid, bromamines, and hypoiodous acid, indicating that R19-S responds to these reactive halogen species as well as HOCl. Hypothiocyanous acid and taurine chloramine were unreactive, however, and ammonia chloramine and dichloramine reacted only very slowly. MS analyses revealed additional products from the reaction of HOCl with R19-S, including a chlorinated species as a minor product. Of note, phagocytosis of opsonized zymosan or Staphylococcus aureus by neutrophils was accompanied by an increase in R19 fluorescence. This increase depended on NADPH oxidase and myeloperoxidase activities, and detection of chlorinated R19-S confirmed its specificity for HOCl. Using live-cell imaging to track individual phagosomes in single neutrophils, we observed considerable heterogeneity among the phagosomes in the time from ingestion of a zymosan particle to when fluorescence was first detected, ranging from 1 to >30 min. However, once initiated, the subsequent fluorescence increase was uniform, reaching a similar maximum in â¼10 min. Our results confirm the utility of R19-S for detecting HOCl in real-time and provide definitive evidence that isolated neutrophils produce HOCl in phagosomes. The intriguing variability in the onset of HOCl production among phagosomes identified here could influence the way they kill ingested bacteria.
Asunto(s)
Colorantes Fluorescentes/química , Ácido Hipocloroso/análisis , Neutrófilos/enzimología , Fagocitosis , Fagosomas/metabolismo , Rodaminas/química , Bioensayo , Colorantes Fluorescentes/metabolismo , Humanos , Ácido Hipocloroso/inmunología , Ácido Hipocloroso/metabolismo , Errores Innatos del Metabolismo/enzimología , Errores Innatos del Metabolismo/inmunología , Errores Innatos del Metabolismo/patología , NADPH Oxidasas/genética , NADPH Oxidasas/inmunología , NADPH Oxidasas/metabolismo , Neutrófilos/inmunología , Neutrófilos/patología , Proteínas Opsoninas/química , Peroxidasa/deficiencia , Peroxidasa/genética , Peroxidasa/inmunología , Fagosomas/inmunología , Fagosomas/ultraestructura , Cultivo Primario de Células , Rodaminas/metabolismo , Espectrometría de Fluorescencia , Staphylococcus aureus/inmunología , Zimosan/químicaRESUMEN
Renal fibrosis is the pathological hallmark of chronic kidney disease (CKD) and manifests as glomerulosclerosis and tubulointerstitial fibrosis. Reactive oxygen species contribute significantly to renal inflammation and fibrosis, but most research has focused on superoxide and hydrogen peroxide (H2O2). The animal heme peroxidases myeloperoxidase (MPO), eosinophil peroxidase (EPX), and peroxidasin (PXDN) uniquely metabolize H2O2 into highly reactive and destructive hypohalous acids, such as hypobromous and hypochlorous acid. However, the role of these peroxidases and their downstream hypohalous acids in the pathogenesis of renal fibrosis is unclear. Our study defines the contribution of MPO, EPX, and PXDN to renal inflammation and tubulointerstitial fibrosis in the murine unilateral ureteral obstruction (UUO) model. Using a nonspecific inhibitor of animal heme peroxidases and peroxidase-specific knockout mice, we find that loss of EPX or PXDN, but not MPO, reduces renal fibrosis. Furthermore, we demonstrate that eosinophils, the source of EPX, accumulate in the renal interstitium after UUO. These findings point to EPX and PXDN as potential therapeutic targets for renal fibrosis and CKD and suggest that eosinophils modulate the response to renal injury.
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Peroxidasa del Eosinófilo/metabolismo , Eosinófilos/enzimología , Proteínas de la Matriz Extracelular/metabolismo , Riñón/enzimología , Nefritis Intersticial/enzimología , Peroxidasa/metabolismo , Peroxidasas/metabolismo , Obstrucción Ureteral/enzimología , Animales , Movimiento Celular , Modelos Animales de Enfermedad , Peroxidasa del Eosinófilo/deficiencia , Peroxidasa del Eosinófilo/genética , Eosinófilos/patología , Proteínas de la Matriz Extracelular/deficiencia , Proteínas de la Matriz Extracelular/genética , Femenino , Fibrosis , Riñón/patología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Nefritis Intersticial/etiología , Nefritis Intersticial/patología , Nefritis Intersticial/prevención & control , Peroxidasa/deficiencia , Peroxidasa/genética , Peroxidasas/deficiencia , Peroxidasas/genética , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Obstrucción Ureteral/complicaciones , Obstrucción Ureteral/patología , PeroxidasinaRESUMEN
RATIONALE: Ventricular arrhythmias remain the leading cause of death in patients suffering myocardial ischemia. Myeloperoxidase, a heme enzyme released by polymorphonuclear neutrophils, accumulates within ischemic myocardium and has been linked to adverse left ventricular remodeling. OBJECTIVE: To reveal the role of myeloperoxidase for the development of ventricular arrhythmias. METHODS AND RESULTS: In different murine models of myocardial ischemia, myeloperoxidase deficiency profoundly decreased vulnerability for ventricular tachycardia on programmed right ventricular and burst stimulation and spontaneously as assessed by ECG telemetry after isoproterenol injection. Experiments using CD11b/CD18 integrin-deficient (CD11b-/-) mice and intravenous myeloperoxidase infusion revealed that neutrophil infiltration is a prerequisite for myocardial myeloperoxidase accumulation. Ventricles from myeloperoxidase-deficient (Mpo-/-) mice showed less pronounced slowing and decreased heterogeneity of electric conduction in the peri-infarct zone than wild-type mice. Expression of the redox-sensitive gap junctional protein Cx43 (Connexin 43) was reduced in the peri-infarct area of wild-type compared with Mpo-/- mice. In isolated wild-type cardiomyocytes, Cx43 protein content decreased on myeloperoxidase/H2O2 incubation. Mapping of induced pluripotent stem cell-derived cardiomyocyte networks and in vivo investigations linked Cx43 breakdown to myeloperoxidase-dependent activation of matrix metalloproteinase 7. Moreover, Mpo-/- mice showed decreased ventricular postischemic fibrosis reflecting reduced accumulation of myofibroblasts. Ex vivo, myeloperoxidase was demonstrated to induce fibroblast-to-myofibroblast transdifferentiation by activation of p38 mitogen-activated protein kinases resulting in upregulated collagen generation. In support of our experimental findings, baseline myeloperoxidase plasma levels were independently associated with a history of ventricular arrhythmias, sudden cardiac death, or implantable cardioverter-defibrillator implantation in a cohort of 2622 stable patients with an ejection fraction >35% undergoing elective diagnostic cardiac evaluation. CONCLUSIONS: Myeloperoxidase emerges as a crucial mediator of postischemic myocardial remodeling and may evolve as a novel pharmacological target for secondary disease prevention after myocardial ischemia.
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Arritmias Cardíacas/metabolismo , Isquemia Miocárdica/metabolismo , Miocitos Cardíacos/metabolismo , Peroxidasa/deficiencia , Remodelación Ventricular/fisiología , Animales , Arritmias Cardíacas/patología , Células Cultivadas , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Isquemia Miocárdica/patología , Miocitos Cardíacos/patología , Técnicas de Cultivo de ÓrganosRESUMEN
Objective- The leukocyte heme-enzyme MPO (myeloperoxidase) exerts proinflammatory effects on the vascular system primarily linked to its catalytic properties. Recent studies have shown that MPO, depending on its cationic charge, mediates neutrophil recruitment and activation. Here, we further investigated MPO's extracatalytic properties and its effect on endothelial glycocalyx (EG) integrity. Approach and Results- In vivo staining of murine cremaster muscle vessels with Alcian Blue 8GX provided evidence of an MPO-dependent decrease in anionic charge of the EG. MPO binding to the glycocalyx was further characterized using Chinese hamster ovary cells and its glycosaminoglycan mutants-pgsA-745 (mutant Chinese hamster ovary cells lacking heparan sulfate and chondroitin sulfate glycosaminoglycan) and pgsD-677 (mutant Chinese hamster ovary cells lacking heparan sulfate glycosaminoglycan), which revealed heparan sulfate as the main mediator of MPO binding. Further, EG integrity was assessed in terms of thickness using intravital microscopy of murine cremaster muscle. A significant reduction in EG thickness was observed on infusion of catalytically active MPO, as well as mutant inactive MPO and cationic polymer polylysine. Similar effects were also observed in wild-type mice after a local inflammatory stimulus but not in MPO-knockout mice. The reduction in EG thickness was reversed after removal of vessel-bound MPO, suggesting a possible physical collapse of the EG. Last, experiments with in vivo neutrophil depletion revealed that MPO also induced neutrophil-mediated shedding of the EG core protein, Sdc1 (syndecan-1). Conclusions- These findings provide evidence that MPO, via ionic interaction with heparan sulfate side chains, can cause neutrophil-dependent Sdc1 shedding and collapse of the EG structure.
Asunto(s)
Músculos Abdominales/irrigación sanguínea , Células Endoteliales/efectos de los fármacos , Glicocálix/efectos de los fármacos , Peroxidasa/metabolismo , Animales , Células CHO , Cationes , Cricetulus , Células Endoteliales/metabolismo , Células Endoteliales/patología , Glicocálix/metabolismo , Glicocálix/patología , Proteoglicanos de Heparán Sulfato/metabolismo , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/patología , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Activación Neutrófila , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Peroxidasa/deficiencia , Peroxidasa/genética , Peroxidasa/farmacología , Unión Proteica , Sindecano-1/metabolismoRESUMEN
Basement membranes (BMs), a specialized form of extracellular matrix, underlie nearly all cell layers and provide structural support for tissues and interact with cell surface receptors to determine cell behavior. Both macromolecular composition and stiffness of the BM influence cell-BM interactions. Collagen IV is a major constituent of the BM that forms an extensively cross-linked oligomeric network. Its deficiency leads to BM mechanical instability, as observed with glomerular BM in Alport syndrome. These findings have led to the hypothesis that collagen IV and its cross-links determine BM stiffness. A sulfilimine bond (S = N) between a methionine sulfur and a lysine nitrogen cross-links collagen IV and is formed by the matrix enzyme peroxidasin. In peroxidasin knockout mice with reduced collagen IV sulfilimine cross-links, we find a reduction in renal tubular BM stiffness. Thus this work provides the first direct experimental evidence that collagen IV sulfilimine cross-links contribute to BM mechanical properties and provides a foundation for future work on the relationship of BM mechanics to cell function in renal disease.
Asunto(s)
Membrana Basal/metabolismo , Colágeno Tipo IV/metabolismo , Reactivos de Enlaces Cruzados/metabolismo , Iminas/metabolismo , Riñón/metabolismo , Animales , Membrana Basal/patología , Fenómenos Biomecánicos , Colágeno Tipo IV/química , Reactivos de Enlaces Cruzados/química , Módulo de Elasticidad , Proteínas de la Matriz Extracelular/deficiencia , Proteínas de la Matriz Extracelular/genética , Genotipo , Iminas/química , Riñón/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Peroxidasa/deficiencia , Peroxidasa/genética , Fenotipo , Conformación Proteica , Resistencia a la Tracción , PeroxidasinaRESUMEN
Oxidative stress plays a fundamental role in abdominal aortic aneurysm (AAA) formation. Activated polymorphonuclear leukocytes (or neutrophils) are associated with AAA and express myeloperoxidase (MPO), which promotes inflammation, matrix degradation, and other pathological features of AAA, including enhanced oxidative stress through generation of reactive oxygen species. Both plasma and aortic MPO levels are elevated in patients with AAA, but the role of MPO in AAA pathogenesis has, heretofore, never been investigated. Here, we show that MPO gene deletion attenuates AAA formation in two animal models: ANG II infusion in apolipoprotein E-deficient mice and elastase perfusion in C57BL/6 mice. Oral administration of taurine [1% or 4% (wt/vol) in drinking water], an amino acid known to react rapidly with MPO-generated oxidants like hypochlorous acid, also prevented AAA formation in the ANG II and elastase models as well as the CaCl2 application model of AAA formation while reducing aortic peroxidase activity and aortic protein-bound dityrosine levels, an oxidative cross link formed by MPO. Both MPO gene deletion and taurine supplementation blunted aortic macrophage accumulation, elastin fragmentation, and matrix metalloproteinase activation, key features of AAA pathogenesis. Moreover, MPO gene deletion and taurine administration significantly attenuated the induction of serum amyloid A, which promotes ANG II-induced AAAs. These data implicate MPO in AAA pathogenesis and suggest that studies exploring whether taurine can serve as a potential therapeutic for the prevention or treatment of AAA in patients merit consideration.NEW & NOTEWORTHY Neutrophils are abundant in abdominal aortic aneurysm (AAA), and myeloperoxidase (MPO), prominently expressed in neutrophils, is associated with AAA in humans. This study demonstrates that MPO gene deletion or supplementation with the natural product taurine, which can scavenge MPO-generated oxidants, can prevent AAA formation, suggesting an attractive potential therapeutic strategy for AAA.
Asunto(s)
Antioxidantes/farmacología , Aorta Abdominal/efectos de los fármacos , Aneurisma de la Aorta Abdominal/prevención & control , Neutrófilos/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Peroxidasa/metabolismo , Taurina/farmacología , Angiotensina II , Animales , Aorta Abdominal/enzimología , Aorta Abdominal/patología , Aneurisma de la Aorta Abdominal/inducido químicamente , Aneurisma de la Aorta Abdominal/enzimología , Aneurisma de la Aorta Abdominal/genética , Cloruro de Calcio , Modelos Animales de Enfermedad , Eliminación de Gen , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados para ApoE , Neutrófilos/enzimología , Elastasa Pancreática , Peroxidasa/deficiencia , Peroxidasa/genética , Especies Reactivas de Oxígeno/metabolismo , Proteína Amiloide A Sérica/metabolismoRESUMEN
OBJECTIVES: Pentraxin 3 (PTX3) is a multifunctional soluble factor. PTX3 can be involved in the regulation of vasculitis and is expressed in the cytoplasm of neutrophils. As anti-neutrophil cytoplasmic antibody (ANCA) is recognised as a cause of vasculitis, we aimed to discover the role of PTX3 in ANCA production in vivo. METHODS: To this end, we used aluminum salt (alum), which induces neutrophil extracellular traps, as an adjuvant for producing anti-myeloperoxidase-ANCA (MPO-ANCA). Specifically, we intraperitoneally injected alum and recombinant MPO (rMPO) into MPO-deficient mice and then measured the concentration of anti-MPO IgG in their blood. To show the involvement of extracellular PTX3 in this model, we assessed PTX3 protein content and host double-stranded DNA levels in the mice's peritoneal fluid after alum injection. In addition, we simultaneously administered recombinant PTX3, rMPO and alum to MPO-deficient mice to assess the function of PTX3 in producing anti-MPO IgG in vivo. RESULTS: Anti-MPO IgG was produced by the alum + rMPO immunisation model in MPO-deficient but not wildtype mice. Injection of alum induced extracellular PTX3 as well as double-stranded DNA and dead cells in MPO-deficient mice. Simultaneous injection of recombinant PTX3 with rMPO and alum attenuated the production of anti-MPO IgG in MPO-deficient mice. CONCLUSIONS: Our current findings provide evidence that PTX3 attenuates the production of murine MPO-ANCA.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Compuestos de Alumbre/farmacología , Anticuerpos Anticitoplasma de Neutrófilos/sangre , Proteína C-Reactiva/inmunología , Inmunoglobulina G/sangre , Errores Innatos del Metabolismo/inmunología , Proteínas del Tejido Nervioso/inmunología , Peroxidasa/inmunología , Animales , Líquido Ascítico/inmunología , Líquido Ascítico/metabolismo , Proteína C-Reactiva/administración & dosificación , Proteína C-Reactiva/metabolismo , ADN/inmunología , ADN/metabolismo , Trampas Extracelulares/inmunología , Trampas Extracelulares/metabolismo , Femenino , Masculino , Errores Innatos del Metabolismo/sangre , Errores Innatos del Metabolismo/enzimología , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas del Tejido Nervioso/administración & dosificación , Proteínas del Tejido Nervioso/metabolismo , Peroxidasa/administración & dosificación , Peroxidasa/deficiencia , Peroxidasa/genéticaRESUMEN
Myeloperoxidase is a key component of neutrophil granules involved in killing engulfed microorganisms. We obtained a zebrafish mutant (smu681) lacking Sudan black staining by large-scale screening, which was a neutrophil-replete but myeloperoxidase-deficient mutant. When infiltrated with Candida albicans, smu681 embryos and sibling embryos showed similar survival after infection. Proliferation of C. albicans was more rapid in smu681 embryos than in sibling embryos, although it was eventually suppressed. In addition, the number of neutrophils accumulating at the site of infection was significantly larger in mutant embryos than in sibling embryos, and mutant embryos showed increased expression of several inflammatory cytokines after C. albicans infection. These findings indicate that myeloperoxidase deficiency alters the inflammatory response to fungal infection.
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Candida albicans/fisiología , Proteínas de Peces/genética , Inmunidad Innata , Peroxidasa/genética , Pez Cebra/inmunología , Pez Cebra/microbiología , Animales , Candida albicans/inmunología , Modelos Animales de Enfermedad , Proteínas de Peces/deficiencia , Proteínas de Peces/metabolismo , Mutación , Peroxidasa/deficiencia , Peroxidasa/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
Necrotizing and crescentic GN (NCGN) with a paucity of glomerular immunoglobulin deposits is associated with ANCA. The most common ANCA target antigens are myeloperoxidase (MPO) and proteinase 3. In a manner that requires activation of the alternative complement pathway, passive transfer of antibodies to mouse MPO (anti-MPO) induces a mouse model of ANCA NCGN that closely mimics human disease. Here, we confirm the importance of C5aR/CD88 in the mediation of anti-MPO-induced NCGN and report that C6 is not required. We further demonstrate that deficiency of C5a-like receptor (C5L2) has the reverse effect of C5aR/CD88 deficiency and results in more severe disease, indicating that C5aR/CD88 engagement enhances inflammation and C5L2 engagement suppresses inflammation. Oral administration of CCX168, a small molecule antagonist of human C5aR/CD88, ameliorated anti-MPO-induced NCGN in mice expressing human C5aR/CD88. These observations suggest that blockade of C5aR/CD88 might have therapeutic benefit in patients with ANCA-associated vasculitis and GN.
Asunto(s)
Vasculitis Asociada a Anticuerpos Citoplasmáticos Antineutrófilos/prevención & control , Autoantígenos/inmunología , Glomerulonefritis/prevención & control , Peroxidasa/inmunología , Receptor de Anafilatoxina C5a/antagonistas & inhibidores , Administración Oral , Animales , Complemento C6/inmunología , Vía Alternativa del Complemento , Relación Dosis-Respuesta a Droga , Técnicas de Sustitución del Gen , Glomerulonefritis/complicaciones , Glomerulonefritis/inmunología , Hematuria/etiología , Hematuria/prevención & control , Humanos , Inmunización Pasiva , Leucocitos , Errores Innatos del Metabolismo/complicaciones , Errores Innatos del Metabolismo/inmunología , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Noqueados , Peroxidasa/deficiencia , Proteinuria/etiología , Proteinuria/prevención & control , Receptor de Anafilatoxina C5a/deficiencia , Receptor de Anafilatoxina C5a/genética , Receptores de Quimiocina/deficiencia , Receptores de Quimiocina/genética , Receptores de Quimiocina/fisiología , Proteínas Recombinantes de Fusión , Orina/citologíaRESUMEN
Autoimmunity to the neutrophil enzyme myeloperoxidase (MPO) is an important cause of rapidly progressive glomerulonephritis, but the relative roles of MPO-specific anti-neutrophil cytoplasmic antibodies (MPO-ANCA) and autoreactive effector MPO-specific CD4(+) T cells are unclear. We confirmed that passive transfer of murine MPO-ANCA to agammaglobulinemic µMT mice immunized with OVA induces glomerular injury with capillary wall thickening, fibrinoid necrosis, mesangial cell proliferation, and periglomerular cell infiltration. Preimmunization of µMT mice with MPO induced MPO-specific CD4(+) T cells and significantly enhanced renal injury after MPO-ANCA transfer. CD4(+) T cell depletion prevented this augmentation of injury, confirming the importance of effector T cells in the development of MPO-ANCA associated glomerulonephritis. Therefore, MPO-ANCA can induce glomerulonephritis through both direct humoral mechanisms (recruitment of neutrophils and deposition of MPO) and indirectly by initiating MPO deposition in glomeruli, thereby directing effector CD4(+) T cell mediated injury. To confirm and support this data, we transferred T cells from MPO-immunized Mpo(-/-)µMT mice into Rag1(-/-) mice (control mice received ovalbumin specific T cells) and triggered injury by passive MPO-ANCA. Renal injury was significantly greater in mice transferred with T cells from MPO-immunized mice. These current studies demonstrate that MPO-ANCA induces injury via both humoral and cell mediated immune mechanisms.
Asunto(s)
Anticuerpos Anticitoplasma de Neutrófilos/inmunología , Linfocitos T CD4-Positivos/inmunología , Glomerulonefritis/inmunología , Peroxidasa/inmunología , Animales , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/trasplante , Femenino , Citometría de Flujo , Glomerulonefritis/genética , Glomerulonefritis/metabolismo , Inmunización , Inmunoglobulina M/genética , Inmunoglobulina M/inmunología , Inmunoglobulina M/metabolismo , Cadenas mu de Inmunoglobulina/genética , Cadenas mu de Inmunoglobulina/inmunología , Glomérulos Renales/inmunología , Glomérulos Renales/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neutrófilos/inmunología , Neutrófilos/metabolismo , Ovalbúmina/inmunología , Peroxidasa/deficiencia , Peroxidasa/genéticaRESUMEN
The pathogenesis of anti-neutrophil cytoplasmic antibody (ANCA)-associated necrotizing crescentic GN (NCGN) is incompletely understood. Dipeptidyl peptidase I (DPPI) is a cysteine protease required for the activation of neutrophil serine proteases (NSPs) cathepsin G, neutrophil elastase, and proteinase 3, which are enzymes that modulate inflammation. We used a mouse model of anti-myeloperoxidase (MPO) antibody-induced NCGN to determine whether active NSPs contribute to its pathogenesis. MPO-deficient animals immunized with murine MPO, irradiated, and transplanted with wild-type bone marrow developed NCGN. In contrast, transplantation with bone marrow that lacked DPPI or lacked both neutrophil elastase and proteinase 3 protected mice from NCGN induced by anti-MPO antibody. The kidneys of mice reconstituted with DPPI-deficient bone marrow generated significantly less IL-1ß than did those of mice reconstituted with wild-type bone marrow; similarly, in vitro, DPPI-deficient monocytes produced significantly less IL-1ß in response to anti-MPO antibody than did wild-type monocytes. This reduction in IL-1ß was NSP dependent; exogenous addition of PR3 restored IL-ß production in DPPI-deficient monocytes. Last, the IL-1 receptor antagonist anakinra protected animals against anti-MPO antibody-induced NCGN (16.7%±6.0% versus 2.4%±1.7% crescents), suggesting that IL-1ß is a critical inflammatory mediator in this model. These data suggest that the development of anti-MPO antibody-induced NCGN requires NSP-dependent IL-1ß generation and that these processes may provide therapeutic targets for ANCA-mediated diseases in humans.
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Glomerulonefritis/metabolismo , Interleucina-1beta/metabolismo , Necrosis de la Corteza Renal/metabolismo , Riñón/metabolismo , Riñón/patología , Neutrófilos/metabolismo , Serina Proteasas/metabolismo , Animales , Anticuerpos Antiidiotipos/efectos adversos , Anticuerpos Anticitoplasma de Neutrófilos/efectos adversos , Trasplante de Médula Ósea , Catepsina C/genética , Catepsina C/metabolismo , Movimiento Celular , Modelos Animales de Enfermedad , Femenino , Glomerulonefritis/inducido químicamente , Glomerulonefritis/patología , Humanos , Proteína Antagonista del Receptor de Interleucina 1/farmacología , Necrosis de la Corteza Renal/inducido químicamente , Necrosis de la Corteza Renal/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Monocitos/metabolismo , Monocitos/patología , Neutrófilos/patología , Peroxidasa/deficiencia , Peroxidasa/genética , Peroxidasa/inmunología , Receptores de Interleucina-1/antagonistas & inhibidores , Receptores de Interleucina-1/efectos de los fármacos , Serina Proteasas/deficienciaRESUMEN
AIMS: Observational studies have suggested a mechanistic link between the leucocyte-derived enzyme myeloperoxidase (MPO) and vasomotor function. Here, we tested whether MPO is systemically affecting vascular tone in humans. METHODS AND RESULTS: A total of 12 135 patients were screened for leucocyte peroxidase activity. We identified 15 individuals with low MPO expression and activity (MPO(low)), who were matched with 30 participants exhibiting normal MPO protein content and activity (control). Nicotine-dependent activation of leucocytes caused attenuation of endothelial nitric oxide (NO) bioavailability in the control group (P < 0.01), but not in MPO(low) individuals (P = 0.12); here the MPO burden of leucocytes correlated with the degree of vasomotor dysfunction (P = 0.008). To directly test the vasoactive properties of free circulating MPO, the enzyme was injected into the left atrium of anaesthetized, open-chest pigs. Myeloperoxidase plasma levels peaked within minutes and rapidly declined thereafter, reflecting vascular binding of MPO. Blood flow in the left anterior descending artery and the internal mammary artery (IMA) as well as myocardial perfusion decreased following MPO injection when compared with albumin-treated animals (P < 0.001). Isolated IMA-rings from animals subjected to MPO revealed markedly diminished relaxation in response to acetylcholine (P < 0.01) and nitroglycerine as opposed to controls (P < 0.001). CONCLUSION: Myeloperoxidase elicits profound effects on vascular tone of conductance and resistance vessels in vivo. These findings not only call for revisiting the biological functions of leucocytes as systemic and mobile effectors of vascular tone, but also identify MPO as a critical systemic regulator of vasomotion in humans and thus a potential therapeutic target.
Asunto(s)
Neutrófilos/enzimología , Peroxidasa/deficiencia , Sistema Vasomotor/enzimología , Adulto , Anciano , Animales , Velocidad del Flujo Sanguíneo , Circulación Coronaria/fisiología , Endotelio Vascular/enzimología , Hemodinámica/fisiología , Humanos , Masculino , Arterias Mamarias/fisiología , Metaloproteinasa 9 de la Matriz/metabolismo , Persona de Mediana Edad , Activación Neutrófila , Nicotina/farmacología , Óxido Nítrico/metabolismo , Peroxidasa/metabolismo , Peroxidasa/farmacología , Sus scrofa , Vasodilatación/fisiología , Adulto JovenRESUMEN
BACKGROUND: The nitric oxide synthase inhibitor asymmetrical dimethylarginine (ADMA) and the leukocyte-derived hemoprotein myeloperoxidase (MPO) are associated with cardiovascular diseases. Activation of monocytes and polymorphonuclear neutrophils (PMNs) with concomitant release of MPO is regulated in a nitric oxide-dependent fashion. The aim of the study was to investigate a potential 2-way interaction between ADMA and MPO. METHODS AND RESULTS: Ex vivo, ADMA uptake by isolated human PMNs, the principal source of MPO in humans, significantly impaired nitric oxide synthase activity determined by gas chromatography-mass spectrometry. In humans, short-term ADMA infusion (0.0125 mg · kg(-1) · min(-1)) significantly increased MPO plasma concentrations. Functionally, PMN exposure to ADMA enhanced leukocyte adhesion to endothelial cells, augmented NADPH oxidase activity, and stimulated PMN degranulation, resulting in release of MPO. In vivo, a 28-day ADMA infusion (250 µmol · kg(-1) · d(-1)) in C57Bl/6 mice significantly increased plasma MPO concentrations, whereas this ADMA effect on MPO was attenuated by human dimethylarginine dimethylaminohydrolase1 (hDDAH1) overexpression. Moreover, the MPO-derived reactive molecule hypochlorous acid impaired recombinant hDDAH1 activity in vitro. In MPO(-/-) mice, the lipopolysaccharide-induced increase in systemic ADMA concentrations was abrogated. CONCLUSIONS: ADMA profoundly impairs nitric oxide synthesis of PMNs, resulting in increased PMN adhesion to endothelial cells, superoxide generation, and release of MPO. In addition, MPO impairs DDAH1 activity. Our data reveal an ADMA-induced cycle of PMN activation, enhanced MPO release, and subsequent impairment of DDAH1 activity. These findings not only highlight so far unrecognized cytokine-like properties of ADMA but also identify MPO as a regulatory switch for ADMA bioavailability under inflammatory conditions.
Asunto(s)
Arginina/análogos & derivados , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Óxido Nítrico Sintasa de Tipo III/antagonistas & inhibidores , Peroxidasa/metabolismo , Amidohidrolasas/genética , Amidohidrolasas/metabolismo , Animales , Arginina/farmacología , Enfermedades Cardiovasculares/metabolismo , Enfermedades Cardiovasculares/fisiopatología , Relación Dosis-Respuesta a Droga , Femenino , Células HL-60 , Humanos , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Modelos Animales , Neutrófilos/citología , Óxido Nítrico/metabolismo , Peroxidasa/deficiencia , Peroxidasa/genética , Transducción de Señal/fisiología , Superóxidos/metabolismoRESUMEN
Antibodies against neutrophil proteins myeloperoxidase (MPO) and proteinase 3 are thought to cause disease in anti-neutrophil cytoplasmic antibody (ANCA) vasculitis. There have been a number of recent developments in the animal models of ANCA vasculitis in both mice and rats. These include models based on an immune response to MPO generated in MPO-deficient mice, with other models using MPO-sufficient mice and rats. In addition, there is a report of the use of humanized mice where immunodeficient mice have been engrafted with human haematopoietic stem cells and injected with patient ANCA. Antibodies to another protein lysosomal-associated protein-2 have been found in patients with ANCA vasculitis, and evidence from a rat model suggests that they are also pathogenic. These models all have advantages and disadvantages, which are discussed. We also consider what these models have taught us about the pathogenesis of ANCA vasculitis. Experiments using genetically modified mice and pharmacological inhibition have given insights into disease mechanisms and have identified potential therapeutic targets. Toll-like receptor stimulation modifies disease by acting both at the level of tissue injury and in the generation of the autoimmune response. Complement is also potentially important with data to support the role of the alternative pathway and C5a in particular. Intracellular pathways have been examined, with a role showing p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase γ. Serine proteases are now known to contribute to disease by release of interleukin-1ß in ANCA-activated neutrophils and monocytes. Other potential therapies studied in these models include the use of bortezemib and strategies to modify antibody glycosylation.
Asunto(s)
Vasculitis Asociada a Anticuerpos Citoplasmáticos Antineutrófilos/inmunología , Modelos Animales , Animales , Autoantígenos/inmunología , Glomerulonefritis/genética , Glomerulonefritis/inmunología , Humanos , Inmunoglobulina G/inmunología , Proteína 2 de la Membrana Asociada a los Lisosomas/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Mutantes , Ratones SCID , Imitación Molecular , Mieloblastina/deficiencia , Mieloblastina/inmunología , Peroxidasa/deficiencia , Peroxidasa/inmunología , Quimera por Radiación/inmunología , Ratas , Ratas Endogámicas WKY , Transducción de Señal , Especificidad de la Especie , Receptores Toll-Like/inmunologíaRESUMEN
OBJECTIVE AND DESIGN: This study examines the role of myeloperoxidase (MPO), a major constituent of neutrophils that generates hypochlorous acid, in neutrophil recruitment into the zymosan-exposed lung of mice. METHODS: Mice were inoculated intranasally with zymosan. The accumulation of neutrophils and other inflammatory cells within the lung was analyzed by flow cytometry. Macrophage inflammatory protein 2 (MIP-2) expression in the lung was quantified, and the contribution of this chemokine to neutrophil accumulation was examined by intranasal administration of MIP-2 antibody. The cellular sources of MIP-2 were identified, and the production of this chemokine from macrophages and neutrophils was quantified in vitro. RESULTS: Zymosan exposure led to greater neutrophil infiltration into the lungs of MPO(-/-) mice relative to wild-type mice. This was associated with higher MIP-2 levels in the mutant mice. Neutralization of MIP-2 in vivo significantly reduced neutrophil infiltration. Neutrophils from MPO(-/-) mice produced more MIP-2, and the production was reduced when MPO was added exogenously. CONCLUSIONS: MPO deficiency results in severe lung inflammation in mice exposed to zymosan. Relatively high MIP-2 levels likely contribute to the strong inflammatory response in these animals.
Asunto(s)
Quimiocina CXCL2/inmunología , Neutrófilos/inmunología , Peroxidasa/inmunología , Neumonía/inmunología , Animales , Líquido del Lavado Bronquioalveolar/citología , Recuento de Células , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Peroxidasa/deficiencia , Peroxidasa/genética , Neumonía/inducido químicamente , Neumonía/patología , ZimosanRESUMEN
Because the pathogenesis of acute respiratory distress syndrome (ARDS) induced by influenza virus infection remains unknown, we can only improve on existing therapeutic interventions. To approach the subject, we investigated immunological etiology focused on cytokines and an acute lung damage factor in influenza-induced ARDS by using a PR-8 (A/H1N1)-infected mouse model. The infected mouse showed fulminant severe pneumonia with leukocyte infiltration, claudin alteration on tight junctions, and formation of hyaline membranes. In addition to interferon (IFN)-α, plenty of keratinocyte-derived chemokines (KC), macrophage inflammatory protein 2 (MIP-2), regulated on activation normal T-cell expressed and secreted (RANTES), and monocyte chemotactic protein 1 (MCP-1) were significantly released into bronchoalveolar lavage fluid (BALF) of the model. We focused on neutrophil myeloperoxidase (MPO) as a potent tissue damage factor and examined its contribution in influenza pneumonia by using mice genetically lacking in MPO. The absence of MPO reduced inflammatory damage with suppression of leakage of total BALF proteins associated with alteration of claudins in the lung. MPO(-/-) mice also suppressed viral load in the lung. The present study suggests that MPO-mediated OCl(-) generation affects claudin molecules and leads to protein leakage and viral spread as a damage factor in influenza-induced ARDS.
Asunto(s)
Subtipo H1N1 del Virus de la Influenza A/patogenicidad , Neutrófilos/inmunología , Infecciones por Orthomyxoviridae/patología , Peroxidasa/metabolismo , Síndrome de Dificultad Respiratoria/patología , Animales , Citocinas/metabolismo , Femenino , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Peroxidasa/deficiencia , Neumonía Viral/patologíaRESUMEN
Polymorphonuclear neutrophils (PMNs) play a key role in host defense. However, their massive accumulation at the site of inflammation can delay regenerative healing processes and can initiate pathological inflammatory processes. Thus, the efficient clearance of PMNs mediated by the induction of regulated cell death is a key process preventing the development of these pathological conditions. Myeloperoxidase (MPO), a highly abundant enzyme in PMN granules, primarily connected with PMN defense machinery, is suggested to play a role in PMN-regulated cell death. However, the contribution of MPO to the mechanisms of PMN cell death remains incompletely characterized. Herein, the process of the cell death of mouse PMNs induced by three different stimuli - phorbol 12-myristate 13-acetate (PMA), opsonized streptococcus (OST), and N-formyl-met-leu-phe (fMLP) - was investigated. MPO-deficient PMNs revealed a significantly decreased rate of cell death characterized by phosphatidylserine surface exposure and cell membrane permeabilization. An inhibitor of MPO activity, 4-aminobenzoic acid hydrazide, did not exhibit a significant effect on PMA-induced cell death compared to MPO deficiency. Interestingly, only the limited activation of markers related to apoptotic cell death was observed (e.g. caspase 8 activation, Bax expression) and they mostly did not correspond to phosphatidylserine surface exposure. Furthermore, a marker characterizing autophagy, cleavage of LC3 protein, as well as histone H3 citrullination and its surface expression was observed. Collectively, the data show the ability of MPO to modulate the life span of PMNs primarily through the potentiation of cell membrane permeabilization and phosphatidylserine surface exposure.
Asunto(s)
Inflamación/metabolismo , Neutrófilos/metabolismo , Peroxidasa/deficiencia , Peroxidasa/metabolismo , Animales , Masculino , Ratones , Ratones Endogámicos C57BL , Neutrófilos/patología , Muerte Celular ReguladaRESUMEN
Generalized pustular psoriasis is a severe psoriatic subtype characterized by epidermal neutrophil infiltration. Although variants in IL36RN and MPO have been shown to affect immune cells, a systematic analysis of neutrophils and PBMC subsets and their differential gene expression dependent on MPO genotypes was not performed yet. We assessed the transcriptomes of MPO-deficient patients using single-cell RNA sequencing of PBMCs and RNA sequencing of neutrophils in a stable disease state. Cell-type annotation by multimodal reference mapping of single-cell RNA-sequencing data was verified by flow cytometry of surface and intracellular markers; the proportions of CD4+ cytotoxic T lymphocytes and other CD4+ effector cells were increased in generalized pustular psoriasis, whereas the frequencies of naïve CD4+ T cells were significantly lower. The expression of FGFBP2 marking CD4+ cytotoxic T lymphocytes and CD8+ effector memory T cells was elevated in patients with generalized pustular psoriasis with disease-contributing variants compared with that in noncarriers (P = 0.0015). In neutrophils, differentially expressed genes were significantly enriched in genes of the classical complement activation pathway. Future studies assessing affected cell types and pathways will show their contribution to generalized pustular psoriasis's pathogenesis and indicate whether findings can be transferred to the acute epidermal situation and whether depletion or inactivation of CD4+ cytotoxic T lymphocytes may be a reasonable therapeutic approach.
Asunto(s)
Peroxidasa , Psoriasis , Enfermedades Cutáneas Vesiculoampollosas , Transcriptoma , Enfermedad Aguda , Linfocitos T CD4-Positivos/patología , Enfermedad Crónica , Humanos , Leucocitos Mononucleares/patología , Peroxidasa/deficiencia , Psoriasis/patología , Enfermedades Cutáneas Vesiculoampollosas/patología , Linfocitos T CitotóxicosRESUMEN
Patients with antineutrophil cytoplasmic antibodies (ANCAs) frequently develop severe vasculitis and glomerulonephritis. Although ANCAs, particularly antimyeloperoxidase (anti-MPO), have been shown to promote leukocyte adhesion in postcapillary venules, their ability to promote adhesion in the glomerular vasculature is less clear. We used intravital microscopy to examine glomerular leukocyte adhesion induced by anti-MPO. In mice pretreated with LPS, 50 microg anti-MPO induced LFA-1-dependent adhesion in glomeruli. In concert with this finding, in mice pretreated with LPS, more than 80% of circulating neutrophils bound anti-MPO within 5 minutes of intravenous administration. However, even in the absence of LPS, more than 40% of circulating neutrophils bound anti-MPO in vivo, a response not seen in MPO(-/-) mice. In addition, a higher dose of anti-MPO (200 microg) induced robust glomerular leukocyte adhesion in the absence of LPS. The latter response was beta2-integrin independent, instead requiring the alpha4-integrin, which was up-regulated on neutrophils in response to anti-MPO. These data indicate that anti-MPO antibodies bind to circulating neutrophils, and can induce glomerular leukocyte adhesion via multiple pathways. Lower doses induce adhesion only after an infection-related stimulus, whereas higher doses are capable of inducing responses in the absence of an additional inflammatory stimulus, via alternative adhesion mechanisms.
Asunto(s)
Anticuerpos Anticitoplasma de Neutrófilos/inmunología , Adhesión Celular/inmunología , Integrina alfa4/fisiología , Glomérulos Renales/irrigación sanguínea , Antígeno-1 Asociado a Función de Linfocito/fisiología , Monocitos/inmunología , Neutrófilos/inmunología , Peroxidasa/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Reacciones Antígeno-Anticuerpo , Antígenos CD18/inmunología , Antígenos CD18/fisiología , Endotoxemia/inmunología , Endotoxinas/farmacología , Endotoxinas/toxicidad , Hidronefrosis/inmunología , Hidronefrosis/patología , Inmunización , Integrina alfa4/metabolismo , Glomérulos Renales/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Monocitos/enzimología , Neutrófilos/enzimología , Selectina-P/inmunología , Peroxidasa/deficiencia , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
Lung neutrophilia is common to a variety of lung diseases. The production of reactive oxygen and nitrogen species during neutrophil oxidative burst has been associated with protein and DNA damage. Myeloperoxidase (MPO) is an enzyme stored in the azurophilic granula of neutrophils. It is important in host defense because it generates the reactive oxidant hypochlorous acid and has been described to play a role in the activation of neutrophils during extravasation. We hypothesized that MPO contributes directly to the development of acute lung neutrophilia via stimulation of neutrophil extravasation and indirectly to the subsequent production of cytokines and chemokines in the lung. To test this hypothesis, wild-type (WT) and Mpo(-/-) mice were given a single LPS instillation, after which the development of neutrophil-dominated lung inflammation, oxidative stress, and cytokine and chemokine levels were examined. Mpo(-/-) mice demonstrated a decreased lung neutrophilia that peaked earlier than neutrophilia in WT mice, which can be explained by decreased neutrophil chemoattractant levels in LPS-exposed Mpo(-/-) compared with WT mice. However, oxidative stress levels were not different in LPS-exposed WT and Mpo(-/-) mice. Furthermore, in vivo findings were confirmed by in vitro studies, using isolated neutrophils. These results indicate that MPO promotes the development of lung neutrophilia and indirectly influences subsequent chemokine and cytokine production by other cell types in the lung.