RESUMEN
Neutrophils are the first leukocytes recruited from the circulation in response to invading pathogens or injured cells. To eradicate pathogens and contribute to tissue repair, recruited neutrophils generate and release a host of toxic chemicals that can also damage normal cells. To avoid collateral damage leading to tissue injury and organ dysfunction, molecular mechanisms evolved that tightly control neutrophil response threshold to activating signals, the strength and location of the response, and the timing of response termination. One mechanism of response control is interruption of activating intracellular signaling pathways by the 20 inhibitory receptors expressed by neutrophils. The two inhibitory C-type lectin receptors expressed by neutrophils, CLEC12A and DCIR, exhibit both common and distinct molecular and functional mechanisms, and they are associated with different diseases. In this review, we use studies on CLEC12A as a model of inhibitory receptor regulation of neutrophil function and participation in disease. Understanding the molecular mechanisms leading to inhibitory receptor specificity offers the possibility of using physiologic control of neutrophil functions as a pharmacologic tool to control inflammatory diseases.
Asunto(s)
Neutrófilos , Transducción de Señal , Humanos , Receptores Mitogénicos/metabolismo , Lectinas Tipo C/metabolismoRESUMEN
BACKGROUND: The mechanisms leading to extracellular matrix (ECM) replacement of areas of glomerular capillaries in histologic variants of FSGS are unknown. This study used proteomics to test the hypothesis that glomerular ECM composition in collapsing FSGS (cFSGS) differs from that of other variants. METHODS: ECM proteins in glomeruli from biopsy specimens of patients with FSGS not otherwise specified (FSGS-NOS) or cFSGS and from normal controls were distinguished and quantified using mass spectrometry, verified and localized using immunohistochemistry (IHC) and confocal microscopy, and assessed for gene expression. The analysis also quantified urinary excretion of ECM proteins and peptides. RESULTS: Of 58 ECM proteins that differed in abundance between cFSGS and FSGS-NOS, 41 were more abundant in cFSGS and 17 in FSGS-NOS. IHC showed that glomerular tuft staining for cathepsin B, cathepsin C, and annexin A3 in cFSGS was significantly greater than in other FSGS variants, in minimal change disease, or in membranous nephropathy. Annexin A3 colocalized with cathepsin B and C, claudin-1, phosphorylated ERK1/2, and CD44, but not with synaptopodin, in parietal epithelial cells (PECs) infiltrating cFSGS glomeruli. Transcripts for cathepsins B and C were increased in FSGS glomeruli compared with normal controls, and urinary excretion of both cathepsins was significantly greater in cFSGS compared with FSGS-NOS. Urinary excretion of ECM-derived peptides was enhanced in cFSGS, although in silico analysis did not identify enhanced excretion of peptides derived from cathepsin B or C. CONCLUSIONS: ECM differences suggest that glomerular sclerosis in cFSGS differs from that in other FSGS variants. Infiltration of activated PECs may disrupt ECM remodeling in cFSGS. These cells and their cathepsins may be therapeutic targets.
Asunto(s)
Proteínas de la Matriz Extracelular/análisis , Glomeruloesclerosis Focal y Segmentaria/metabolismo , Glomérulos Renales/metabolismo , Proteómica/métodos , Catepsinas/fisiología , Células Epiteliales/fisiología , Humanos , Inmunohistoquímica , Glomérulos Renales/química , Microscopía ConfocalRESUMEN
CLEC12A is a myeloid inhibitory receptor that negatively regulates inflammation in mouse models of autoimmune and autoinflammatory arthritis. Reduced CLEC12A expression enhances myeloid cell activation and inflammation in CLEC12A knock-out mice with collagen antibody-induced or gout-like arthritis. Similarly to other C-type lectin receptors, CLEC12A harbours a stalk domain between its ligand binding and transmembrane domains. While it is presumed that the cysteines in the stalk domain have multimerisation properties, their role in CLEC12A expression and/or signaling remain unknown. We thus used site-directed mutagenesis to determine whether the stalk domain cysteines play a role in CLEC12A expression, internalisation, oligomerisation, and/or signaling. Mutation of C118 blocks CLEC12A transport through the secretory pathway diminishing its cell-surface expression. In contrast, mutating C130 does not affect CLEC12A cell-surface expression but increases its oligomerisation, inducing ligand-independent phosphorylation of the receptor. Moreover, we provide evidence that CLEC12A dimerisation is regulated in a redox-dependent manner. We also show that antibody-induced CLEC12A cross-linking induces flotillin oligomerisation in insoluble membrane domains in which CLEC12A signals. Taken together, these data indicate that the stalk cysteines in CLEC12A differentially modulate this inhibitory receptor's expression, oligomerisation and signaling, suggestive of the regulation of CLEC12A in a redox-dependent manner during inflammation.
Asunto(s)
Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Proteínas de la Membrana/metabolismo , Células Mieloides/metabolismo , Multimerización de Proteína/genética , Receptores Mitogénicos/genética , Receptores Mitogénicos/metabolismo , Línea Celular Tumoral , Cisteína/metabolismo , Células HEK293 , Células HeLa , Humanos , Inflamación/genética , Lectinas Tipo C/biosíntesis , Proteínas de la Membrana/genética , Mutagénesis Sitio-Dirigida , Fosforilación , Dominios Proteicos/genética , Transporte de Proteínas/genética , Receptores Mitogénicos/biosíntesis , Transducción de Señal/inmunologíaRESUMEN
Acute glomerulonephritis is characterized by rapid glomerular neutrophil recruitment, proteinuria, and glomerular hypercellularity. The current study tested the hypothesis that the release of neutrophil granule contents plays a role in both the loss of filtration barrier leading to proteinuria and the increase in glomerular cells. Inhibition of neutrophil exocytosis with a peptide inhibitor prevented proteinuria and attenuated podocyte and endothelial cell injury but had no effect on glomerular hypercellularity in an experimental acute glomerulonephritis model in mice. Cultivation of podocytes with neutrophil granule contents disrupted cytoskeletal organization, an in vitro model for podocyte effacement and loss of filtration barrier. Activated, cultured podocytes released cytokines that stimulated neutrophil chemotaxis, primed respiratory burst activity, and stimulated neutrophil exocytosis. We conclude that crosstalk between podocytes and neutrophils contributes to disruption of the glomerular filtration barrier in acute glomerulonephritis. Neutrophil granule products induce podocyte injury but do not participate in the proliferative response of intrinsic glomerular cells.
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Citoesqueleto de Actina/metabolismo , Enfermedad por Anticuerpos Antimembrana Basal Glomerular/metabolismo , Comunicación Celular , Exocitosis , Tasa de Filtración Glomerular , Neutrófilos/metabolismo , Podocitos/metabolismo , Proteinuria/metabolismo , Citoesqueleto de Actina/patología , Animales , Enfermedad por Anticuerpos Antimembrana Basal Glomerular/patología , Enfermedad por Anticuerpos Antimembrana Basal Glomerular/fisiopatología , Enfermedad por Anticuerpos Antimembrana Basal Glomerular/prevención & control , Línea Celular , Citocinas/metabolismo , Modelos Animales de Enfermedad , Exocitosis/efectos de los fármacos , Femenino , Productos del Gen tat/farmacología , Humanos , Masculino , Ratones Endogámicos C57BL , Activación Neutrófila , Infiltración Neutrófila , Neutrófilos/efectos de los fármacos , Podocitos/patología , Proteinuria/patología , Proteinuria/fisiopatología , Proteinuria/prevención & control , Especies Reactivas de Oxígeno/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Estallido Respiratorio , Proteínas SNARE/farmacologíaRESUMEN
Transcription factor NF-κB regulates expression of numerous genes that control inflammation and is activated in glomerular cells in glomerulonephritis (GN). We previously identified genetic variants for a NF-κB regulatory, ubiquitin-binding protein ABIN1 as risk factors for GN in systemic autoimmunity. The goal was to define glomerular inflammatory events controlled by ABIN1 function in GN. Nephrotoxic serum nephritis was induced in wild-type (WT) and ubiquitin-binding deficient ABIN1[D485N] mice, and renal pathophysiology and glomerular inflammatory phenotypes were assessed. Proteinuria was also measured in ABIN1[D485N] mice transplanted with WT mouse bone marrow. Inflammatory activation of ABIN1[D472N] (D485N homolog) cultured human-derived podocytes, and interaction with primary human neutrophils were also assessed. Disruption of ABIN1 function exacerbated proteinuria, podocyte injury, glomerular NF-κB activity, glomerular expression of inflammatory mediators, and glomerular recruitment and retention of neutrophils in antibody-mediated nephritis. Transplantation of WT bone marrow did not prevent the increased proteinuria in ABIN1[D845N] mice. Tumor necrosis factor-stimulated enhanced expression and secretion of NF-κB-targeted proinflammatory mediators in ABIN1[D472N] cultured podocytes compared with WT cells. Supernatants from ABIN1[D472N] podocytes accelerated chemotaxis of human neutrophils, and ABIN1[D472N] podocytes displayed a greater susceptibility to injurious morphologic findings induced by neutrophil granule contents. These studies define a novel role for ABIN1 dysfunction and NF-κB in mediating GN through proinflammatory activation of podocytes.
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Proteínas de Unión al ADN/metabolismo , Glomerulonefritis/patología , FN-kappa B/metabolismo , Podocitos/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Glomerulonefritis/metabolismo , Humanos , Inflamación/metabolismo , Inflamación/patología , Ratones , Ratones MutantesRESUMEN
Ab maturation as well as memory B and plasma cell differentiation occur primarily in the germinal centers (GCs). Systemic lupus erythematosus (SLE) may develop as a result of enhanced GC activity. Previous studies have shown that the dysregulated STAT3 pathway is linked to lupus pathogenesis. However, the exact role of STAT3 in regulating SLE disease progression has not been fully understood. In this study, we demonstrated that STAT3 signaling in B cells is essential for GC formation and maintenance as well as Ab response. Increased cell apoptosis and downregulated Bcl-xL and Mcl-1 antiapoptotic gene expression were found in STAT3-deficient GC B cells. The follicular helper T cell response positively correlated with GC B cells and was significantly decreased in immunized B cell STAT3-deficient mice. STAT3 deficiency also led to the defect of plasma cell differentiation. Furthermore, STAT3 deficiency in autoreactive B cells resulted in decreased autoantibody production. Results obtained from B cell STAT3-deficient B6.MRL/lpr mice suggest that STAT3 signaling significantly contributes to SLE pathogenesis by regulation of GC reactivity, autoantibody production, and kidney pathology. Our findings provide new insights into the role of STAT3 signaling in the maintenance of GC formation and GC B cell differentiation and identify STAT3 as a novel target for treatment of SLE.
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Linfocitos B/inmunología , Linfocitos B/metabolismo , Centro Germinal/citología , Centro Germinal/inmunología , Lupus Eritematoso Sistémico/inmunología , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Animales , Modelos Animales de Enfermedad , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor de Transcripción STAT3/deficienciaRESUMEN
BACKGROUND: Acute kidney injury (AKI) is a common post-cardiac surgery complication and influences patient morbidity and prognosis. This study was designed to identify preoperative candidate urine biomarkers in patients undergoing cardiac surgery. METHODS: A prospective cohort study of adults undergoing cardiac surgery at increased risk for AKI at a single hospital between July 2010 and September 2012 was performed. The primary outcome was the development of AKI, defined as an absolute serum creatinine (SCr) level increase ≥ 0.5 mg/dL or a ≥ 50% relative increase within 72 h of surgery. A secondary outcome was development of AKI defined by Kidney Disease Improving Global Outcomes (KDIGO). Urine collected by voiding within 4 h prior to surgery was used for proteomic analysis and confirmatory enzyme linked immunosorbent assays (ELISAs) studies. Biomarkers were tested for AKI-prediction using Cox and Snell R2, area under the receiver operating curve (AUROC), and percent of corrected classifications. To evaluate the added effect of each candidate biomarker on AKI discrimination, receiver operator characteristic (ROC) curves, integrated discrimination improvement (IDI), and net reclassification improvement (NRI) were calculated. RESULTS: Forty-seven of 755 patients met screening criteria including 15 with AKI. Proteomic analysis identified 29 proteins with a significant ≥2-fold change. Confirmatory ELISA measurements of five candidate markers showed urinary complement factor B (CFB) and histidine rich glycoprotein (HRG) concentrations were significantly increased in patients with AKI. By multivariate analysis, NRI, and IDI the addition of CFB and HRG to the standard clinical assessment significantly improved risk prediction for the primary outcome. Only HRG was a significant predictor in the 21 patients with AKI defined by KDIGO criteria. CONCLUSIONS: Pre-operative urine measurement of CFB or HRG significantly enhanced the current post-surgery AKI risk stratification for more restrictive definition of AKI. HRG, but not CFB or clinical risk stratification, predicted AKI defined by KDIGO. The ability of these biomarkers to predict risk for dialysis-requiring AKI or death could not be reliably assessed in our study due to a small number of patients with either outcome.
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Lesión Renal Aguda/diagnóstico , Lesión Renal Aguda/orina , Procedimientos Quirúrgicos Cardíacos/efectos adversos , Complicaciones Posoperatorias/diagnóstico , Complicaciones Posoperatorias/orina , Lesión Renal Aguda/epidemiología , Anciano , Biomarcadores/orina , Procedimientos Quirúrgicos Cardíacos/tendencias , Estudios de Cohortes , Femenino , Humanos , Masculino , Persona de Mediana Edad , Complicaciones Posoperatorias/epidemiología , Valor Predictivo de las Pruebas , Estudios Prospectivos , Factores de RiesgoRESUMEN
Significant advances in understanding the pathogenesis of GN have occurred in recent decades. Among those advances is the finding that both innate and adaptive immune cells contribute to the development of GN. Neutrophils were recognized as key contributors in early animal models of GN, at a time when the prevailing view considered neutrophils to function as nonspecific effector cells that die quickly after performing antimicrobial functions. However, advances over the past two decades have shown that neutrophil functions are more complex and sophisticated. Specifically, research has revealed that neutrophil survival is regulated by the inflammatory milieu and that neutrophils demonstrate plasticity, mediate microbial killing through previously unrecognized mechanisms, demonstrate transcriptional activity leading to the release of cytokines and chemokines, interact with and regulate cells of the innate and adaptive immune systems, and contribute to the resolution of inflammation. Therefore, neutrophil participation in glomerular diseases deserves re-evaluation. In this review, we describe advances in understanding classic neutrophil functions, review the expanded roles of neutrophils in innate and adaptive immune responses, and summarize current knowledge of neutrophil contributions to GN.
Asunto(s)
Glomerulonefritis/inmunología , Neutrófilos/fisiología , Inmunidad Adaptativa , Animales , Glomerulonefritis/microbiología , Humanos , Inmunidad Innata , Infiltración NeutrófilaRESUMEN
Abnormal extracellular matrix (ECM) remodeling is a prominent feature of many glomerular diseases and is a final common pathway of glomerular injury. However, changes in ECM composition accompanying disease-related remodeling are unknown. The physical properties of ECM create challenges for characterization of composition using standard protein extraction techniques, as the insoluble components of ECM are frequently discarded and many ECM proteins are in low abundance compared to other cell proteins. Prior proteomic studies defining normal ECM composition used a large number of glomeruli isolated from human kidneys retrieved for transplantation or by nephrectomy for cancer. Here we examined the ability to identify ECM proteins by mass spectrometry using glomerular sections compatible with those available from standard renal biopsy specimens. Proteins were classified as ECM by comparison to the Matrisome database and previously identified glomerular ECM proteins. Optimal ECM protein identification resulted from sequential decellularization and protein extraction of 100 human glomerular sections isolated by laser capture microdissection from either frozen or formalin-fixed, paraffin-embedded tissue. In total, 147 ECM proteins were identified, including the majority of structural and GBM proteins previously identified along with a number of matrix and glomerular basement membrane proteins not previously associated with glomeruli. Thus, our study demonstrates the feasibility of proteomic analysis of glomerular ECM from retrieved glomerular sections isolated from renal biopsy tissue and expands the list of known ECM proteins in glomeruli.
Asunto(s)
Proteínas de la Matriz Extracelular/análisis , Matriz Extracelular/química , Membrana Basal Glomerular/química , Enfermedades Renales/metabolismo , Captura por Microdisección con Láser , Proteómica/métodos , Biomarcadores/análisis , Biopsia , Bases de Datos de Proteínas , Matriz Extracelular/patología , Estudios de Factibilidad , Fijadores , Formaldehído , Secciones por Congelación , Membrana Basal Glomerular/patología , Humanos , Enfermedades Renales/diagnóstico , Espectrometría de Masas , Adhesión en Parafina , Valor Predictivo de las Pruebas , Mapas de Interacción de Proteínas , Reproducibilidad de los Resultados , Fijación del Tejido/métodosRESUMEN
OBJECTIVE AND DESIGN: Neutrophil generation of reactive oxygen species (ROS) is enhanced by exposure to pro-inflammatory agents in a process termed priming. Priming is depending on exocytosis of neutrophil granules and p47phox phosphorylation-dependent translocation of cytosolic NADPH oxidase components. Clathrin-mediated endocytosis was recently reported to be necessary for priming, but the mechanism linking endocytosis to priming was not identified. The present study examined the hypothesis that endocytosis regulates neutrophil priming by controlling granule exocytosis. MATERIALS AND METHODS: Clathrin-mediated endocytosis by isolated human neutrophils was inhibited by chlorpromazine, monodansylcadaverine, and sucrose. Exocytosis of granule subsets was measured as release of granule components by ELISA or chemiluminescence. ROS generation was measured as extracellular release of superoxide as reduction of ferrocytochrome c. p38 MAPK activation and p47phox phosphorylation were measured by immunoblot analysis. Statistical analysis was performed using a one-way ANOVA with the Tukey-Kramer multiple-comparison test. RESULTS: Inhibition of endocytosis prevented priming of superoxide release by TNFα and inhibited TNFα stimulation and priming of exocytosis of all four granule subsets. Inhibition of endocytosis did not reduce TNFα-stimulated p38 MAPK activation or p47phox phosphorylation. Inhibition of NADPH oxidase activity blocked TNFα stimulation of secretory vesicle and gelatinase granule exocytosis. CONCLUSIONS: Endocytosis is linked to priming of respiratory burst activity through ROS-mediated control of granule exocytosis.
Asunto(s)
Endocitosis/fisiología , Exocitosis/fisiología , Neutrófilos/fisiología , Estallido Respiratorio/fisiología , Clatrina/farmacología , Endocitosis/efectos de los fármacos , Exocitosis/efectos de los fármacos , Humanos , NADH NADPH Oxidorreductasas/metabolismo , NADPH Oxidasas/metabolismo , Neutrófilos/efectos de los fármacos , Fosforilación , Especies Reactivas de Oxígeno/metabolismo , Estallido Respiratorio/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
The pore-forming toxin listeriolysin O (LLO) is a major virulence factor secreted by the facultative intracellular pathogen Listeria monocytogenes. This toxin facilitates L. monocytogenes intracellular survival in macrophages and diverse nonphagocytic cells by disrupting the internalization vesicle, releasing the bacterium into its replicative niche, the cytosol. Neutrophils are innate immune cells that play an important role in the control of infections, yet it was unknown if LLO could confer a survival advantage to L. monocytogenes in neutrophils. We report that LLO can enhance the phagocytic efficiency of human neutrophils and is unable to protect L. monocytogenes from intracellular killing. To explain the absence of L. monocytogenes survival in neutrophils, we hypothesized that neutrophil degranulation leads to the release of LLO-neutralizing molecules in the forming phagosome. In support of this, L. monocytogenes is a potent inducer of neutrophil degranulation, since its virulence factors, such as LLO, facilitate granule exocytosis. Within the first few minutes of interaction with L. monocytogenes, granules can fuse with the plasma membrane at the bacterial interaction site before closure of the phagosome. Furthermore, granule products directly degrade LLO, irreversibly inhibiting its activity. The matrix metalloproteinase-8, stored in secondary granules, was identified as an endoprotease that degrades LLO, and blocking neutrophil proteases increased L. monocytogenes intracellular survival. In conclusion, we propose that LLO degradation by matrix metalloproteinase-8 during phagocytosis protects neutrophil membranes from perforation and contributes to maintaining L. monocytogenes in a bactericidal phagosome from which it cannot escape.
Asunto(s)
Toxinas Bacterianas/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas Hemolisinas/metabolismo , Listeria monocytogenes/inmunología , Listeria monocytogenes/metabolismo , Metaloproteinasa 8 de la Matriz/metabolismo , Neutrófilos/inmunología , Neutrófilos/metabolismo , Animales , Degranulación de la Célula/inmunología , Línea Celular , Humanos , Macrófagos/inmunología , Macrófagos/microbiología , Ratones , Modelos Inmunológicos , Neutrófilos/microbiología , Fagocitosis/inmunología , Fagosomas/inmunología , Fagosomas/metabolismoRESUMEN
Cell-derived vesicles represent a recently discovered mechanism for intercellular communication. We investigated their potential role in interaction of microbes with host organisms. We provide evidence that different stimuli induced isolated neutrophilic granulocytes to release microvesicles with different biologic properties. Only opsonized particles initiated the formation of microvesicles that were able to impair bacterial growth. The antibacterial effect of neutrophil-derived microvesicles was independent of production of toxic oxygen metabolites and opsonization or engulfment of the microbes, but depended on ß(2) integrin function, continuous actin remodeling, and on the glucose supply. Neutrophil-derived microvesicles were detected in the serum of healthy donors, and their number was significantly increased in the serum of bacteremic patients. We propose a new extracellular mechanism to restrict bacterial growth and dissemination.
Asunto(s)
Bacteriemia/inmunología , Micropartículas Derivadas de Células/inmunología , Vesículas Citoplasmáticas/inmunología , Neutrófilos/inmunología , Neutrófilos/microbiología , Proteínas Opsoninas/metabolismo , Adulto , Bacteriemia/sangre , Micropartículas Derivadas de Células/microbiología , Quimiocina CXCL12/farmacología , Factores Quimiotácticos/farmacología , Vesículas Citoplasmáticas/efectos de los fármacos , Citoesqueleto/fisiología , Humanos , Lipopolisacáridos/farmacología , Neutrófilos/ultraestructura , Proteínas Opsoninas/inmunología , Infecciones Estafilocócicas/inmunología , Staphylococcus aureus/crecimiento & desarrollo , Staphylococcus aureus/inmunologíaRESUMEN
Neutrophils kill bacteria generally through oxidative and nonoxidative mechanisms. Whereas much research has focused on the enzymes essential for neutrophil killing, little is known about the regulatory molecules responsible for such killing. In this study, we investigated the role of olfactomedin 4 (OLFM4), an olfactomedin-related glycoprotein, in neutrophil bactericidal capability and host innate immunity. Neutrophils from OLFM4â»/â» mice have increased intracellular killing of Staphylococcus aureus and Escherichia coli in vitro. The OLFM4â»/â» mice have enhanced in vivo bacterial clearance and are more resistant to sepsis when challenged with S. aureus or E. coli by i.p. injection. OLFM4 was found to interact with cathepsin C, a cysteine protease that plays an important role in bacterial killing and immune regulation. We demonstrated that OLFM4 inhibited cathepsin C activity in vitro and in vivo. The cathepsin C activity in neutrophils from OLFM4â»/â» mice was significantly higher than that in neutrophils from wild-type littermate mice. The activities of three serine proteases (neutrophil elastase, cathepsin G, and proteinase 3), which require cathepsin C activity for processing and maturity, were also significantly higher in OLFM4â»/â» neutrophils. The bacterial killing and clearance capabilities observed in OLFM4â»/â» mice that were enhanced relative to wild-type mice were significantly compromised by the additional loss of cathepsin C in mice with OLFM4 and cathepsin C double deficiency. These results indicate that OLFM4 is an important negative regulator of neutrophil bactericidal activity by restricting cathepsin C activity and its downstream granule-associated serine proteases.
Asunto(s)
Catepsina C/antagonistas & inhibidores , Infecciones por Escherichia coli/inmunología , Factor Estimulante de Colonias de Granulocitos/fisiología , Activación Neutrófila/inmunología , Inhibidores de Proteasas/metabolismo , Infecciones Estafilocócicas/inmunología , Animales , Catepsina C/fisiología , Infecciones por Escherichia coli/enzimología , Infecciones por Escherichia coli/patología , Factor Estimulante de Colonias de Granulocitos/deficiencia , Células HEK293 , Células HeLa , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Infecciones Estafilocócicas/enzimología , Infecciones Estafilocócicas/patologíaRESUMEN
The genetic factors underlying the pathogenesis of lupus nephritis associated with systemic lupus erythematosus are largely unknown, although animal studies indicate that nuclear factor (NF)-κB is involved. We reported previously that a knockin mouse expressing an inactive form of ABIN1 (ABIN1[D485N]) develops lupus-like autoimmune disease and demonstrates enhanced activation of NF-κB and mitogen-activated protein kinases in immune cells after toll-like receptor stimulation. In the current study, we show that ABIN1[D485N] mice develop progressive GN similar to class III and IV lupus nephritis in humans. To investigate the clinical relevance of ABIN1 dysfunction, we genotyped five single-nucleotide polymorphisms in the gene encoding ABIN1, TNIP1, in samples from European-American, African American, Asian, Gullah, and Hispanic participants in the Large Lupus Association Study 2. Comparing cases of systemic lupus erythematosus with nephritis and cases of systemic lupus erythematosus without nephritis revealed strong associations with lupus nephritis at rs7708392 in European Americans and rs4958881 in African Americans. Comparing cases of systemic lupus erythematosus with nephritis and healthy controls revealed a stronger association at rs7708392 in European Americans but not at rs4958881 in African Americans. Our data suggest that variants in the TNIP1 gene are associated with the risk for lupus nephritis and could be mechanistically involved in disease development via aberrant regulation of NF-κB and mitogen-activated protein kinase activity.
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Proteínas de Unión al ADN/fisiología , Nefritis Lúpica/genética , Animales , Proteínas de Unión al ADN/genética , Técnica del Anticuerpo Fluorescente , Humanos , Riñón/patología , Riñón/fisiopatología , Nefritis Lúpica/etiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , FN-kappa B/fisiología , Polimorfismo de Nucleótido SimpleRESUMEN
The role of exocytosis in the human neutrophil respiratory burst was determined using a fusion protein (TAT-SNAP-23) containing the HIV transactivator of transcription (TAT) cell-penetrating sequence and the N-terminal SNARE domain of synaptosome-associated protein-23 (SNAP-23). This agent inhibited stimulated exocytosis of secretory vesicles and gelatinase and specific granules but not azurophil granules. GST pulldown showed that TAT-SNAP-23 bound to the combination of vesicle-associated membrane protein-2 and syntaxin-4 but not to either individually. TAT-SNAP-23 reduced phagocytosis-stimulated hydrogen peroxide production by 60% without affecting phagocytosis or generation of HOCl within phagosomes. TAT-SNAP-23 had no effect on fMLF-stimulated superoxide release but significantly inhibited priming of this response by TNF-α and platelet-activating factor. Pretreatment with TAT-SNAP-23 inhibited the increase in plasma membrane expression of gp91(phox) in TNF-α-primed neutrophils, whereas TNF-α activation of ERK1/2 and p38 MAPK was not affected. The data demonstrate that neutrophil granule exocytosis contributes to phagocytosis-induced respiratory burst activity and plays a critical role in priming of the respiratory burst by increasing expression of membrane components of the NADPH oxidase.
Asunto(s)
Gránulos Citoplasmáticos/inmunología , Exocitosis/inmunología , Activación Neutrófila/inmunología , Estallido Respiratorio/inmunología , Apoptosis/genética , Apoptosis/inmunología , Gránulos Citoplasmáticos/genética , Gránulos Citoplasmáticos/metabolismo , Exocitosis/genética , Productos del Gen tat/antagonistas & inhibidores , Productos del Gen tat/genética , Productos del Gen tat/metabolismo , VIH-1/inmunología , Humanos , Activación Neutrófila/genética , Fagocitosis/genética , Fagocitosis/inmunología , Factor de Activación Plaquetaria/fisiología , Estructura Terciaria de Proteína/genética , Proteínas Qb-SNARE/antagonistas & inhibidores , Proteínas Qb-SNARE/genética , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/antagonistas & inhibidores , Proteínas Qc-SNARE/genética , Proteínas Qc-SNARE/metabolismo , Proteínas Recombinantes de Fusión/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/metabolismo , Estallido Respiratorio/genética , Proteínas SNARE/antagonistas & inhibidores , Proteínas SNARE/genética , Proteínas SNARE/metabolismo , Transducción de Señal/genética , Transducción de Señal/inmunología , Factor de Necrosis Tumoral alfa/fisiologíaRESUMEN
Regulated exocytosis of neutrophil intracellular storage granules is necessary for neutrophil participation in the inflammatory response. The signal transduction pathways that participate in neutrophil exocytosis are complex and poorly defined. Several protein kinases, including p38 MAPK and the nonreceptor tyrosine kinases, Hck and Fgr, participate in this response. However, the downstream targets of these kinases that regulate exocytosis are unknown. The present study combined a novel inhibitor of neutrophil exocytosis with proteomic techniques to identify phosphopeptides and phosphoproteins from a population of gelatinase and specific granules isolated from unstimulated and fMLF-stimulated neutrophils. To prevent loss of granule-associated phosphoproteins upon exocytosis, neutrophils were pretreated with a TAT-fusion protein containing a SNARE domain from SNAP-23 (TAT-SNAP-23), which inhibited fMLF-stimulated CD66b-containing granule exocytosis by 100±10%. Following TAT-SNAP-23 pretreatment, neutrophils were stimulated with the chemotactic peptide fMLF for 0 min, 1 min, and 2 min. Granules were isolated by gradient centrifugation and subjected to proteolytic digestion with trypsin or chymotrypsin to obtain peptides from the outer surface of the granule. Phosphopeptides were enriched by gallium or TiO2 affinity chromatography, and phosphopeptides and phosphorylation sites were identified by reversed phase high performance liquid chromatography-electrospray ionization-tandem MS. This resulted in the identification of 243 unique phosphopeptides corresponding to 235 proteins, including known regulators of vesicle trafficking. The analysis identified 79 phosphoproteins from resting neutrophils, 81 following 1 min of fMLF stimulation, and 118 following 2 min of stimulation. Bioinformatic analysis identified a potential Src tyrosine kinase motif from a phosphopeptide corresponding to G protein coupled receptor kinase 5 (GRK5). Phosphorylation of GRK5 by Src was confirmed by an in vitro kinase reaction and by precursor ion scanning for phospho-tyrosine specific immonium ions containing Tyr251 and Tyr253. Immunoprecipitation of phosphorylated GRK5 from intact cells was reduced by a Src inhibitor. In conclusion, targets of signal transduction pathways were identified that are candidates to regulate neutrophil granule exocytosis.
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Quimiotaxis/efectos de los fármacos , Gránulos Citoplasmáticos/metabolismo , Productos del Gen tat/farmacología , Activación Neutrófila/efectos de los fármacos , Neutrófilos/citología , Neutrófilos/metabolismo , Fosfoproteínas/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Proteínas SNARE/farmacología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Biología Computacional , Gránulos Citoplasmáticos/efectos de los fármacos , Gránulos Citoplasmáticos/enzimología , Exocitosis/efectos de los fármacos , Quinasa 5 del Receptor Acoplado a Proteína-G/metabolismo , Humanos , Datos de Secuencia Molecular , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/efectos de los fármacos , Neutrófilos/enzimología , Fosfopéptidos/química , Fosfopéptidos/metabolismo , Fosfoproteínas/clasificación , Fosforilación/efectos de los fármacos , Proteínas Qb-SNARE/metabolismo , Factores de Tiempo , Proteínas Quinasas p38 Activadas por Mitógenos/química , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Familia-src Quinasas/química , Familia-src Quinasas/metabolismoRESUMEN
AIM: Relatively little is known about the prevalence of acute kidney injury developing outside a hospital setting (CA-AKI) or the impact of CA-AKI on short-term or long-term clinical outcomes. The objective of this study was to compare the prevalence, causes, severity and outcomes of patients with CA-AKI and hospital-acquired (HA)-AKI. METHODS: A retrospective cohort study of patients with AKI identified by ICD-9 code at a single VA (Veterans Affairs) hospital from September 1999 to May 2007 was performed. AKI was verified by applying the RIFLE criteria, and patients were categorized as CA-AKI if RIFLE criteria were met at admission. Demographic, clinical, and outcome variables were extracted by chart review. RESULTS: Four hundred twenty-two patients met inclusion criteria, of which 335 (79.4%) developed CA-AKI. Patients with CA-AKI were more likely to have volume depletion as the aetiology, had fewer chronic illnesses and hospital complications, had a shorter length of stay, and had a reduced mortality, compared with HA-AKI. Distribution among the three RIFLE classes did not differ between groups, and recovery of renal function was incomplete in both groups. CONCLUSION: We conclude that CA-AKI is a common cause of AKI that is as severe as that seen in HA-AKI. CA-AKI has a significant impact on length of hospital stay, mortality, and the development and/or progression of chronic kidney disease. Strategies to limit the risk of CA-AKI are likely to have a significant impact on healthcare costs and patient care.
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Lesión Renal Aguda/epidemiología , Hospitalización , Lesión Renal Aguda/diagnóstico , Lesión Renal Aguda/mortalidad , Lesión Renal Aguda/terapia , Anciano , Anciano de 80 o más Años , Análisis de Varianza , Distribución de Chi-Cuadrado , Progresión de la Enfermedad , Mortalidad Hospitalaria , Hospitales de Veteranos , Humanos , Tiempo de Internación , Persona de Mediana Edad , Prevalencia , Pronóstico , Insuficiencia Renal Crónica/epidemiología , Estudios Retrospectivos , Medición de Riesgo , Factores de Riesgo , Índice de Severidad de la Enfermedad , Factores de Tiempo , Estados Unidos/epidemiología , United States Department of Veterans AffairsRESUMEN
Neutrophils are a specialized subset of white blood cells, which have the ability to store pre-formed mediators in their cytoplasmic granules. Neutrophils are well-known effector cells involved in host protection against pathogens through diverse mechanisms such as phagocytosis, degranulation, extracellular traps, and oxidative burst. In this study, we provide evidence highlighting the significance of the SNARE proteins syntaxin-4 and synaptosomal-associated protein (SNAP) 23 in the release of azurophilic granules, specific granules, and the production of reactive oxygen species in human neutrophils. In contrast, the specific blockade of either syntaxin-4 or SNAP23 did not prevent the release of mitochondrial dsDNA in the process of neutrophil extracellular trap (NET) formation. These findings imply that degranulation and the release of mitochondrial dsDNA involve at least partially distinct molecular pathways in neutrophils.
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Trampas Extracelulares , Proteínas Qa-SNARE , Proteínas Qb-SNARE , Proteínas Qc-SNARE , Humanos , ADN Mitocondrial/metabolismo , Exocitosis , Trampas Extracelulares/metabolismo , Neutrófilos/metabolismo , Proteínas Qa-SNARE/metabolismo , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/metabolismoRESUMEN
Neutrophils play a significant role in determining disease severity following SARS-CoV-2 infection. Gene and protein expression defines several neutrophil clusters in COVID-19, including the emergence of low density neutrophils (LDN) that are associated with severe disease. The functional capabilities of these neutrophil clusters and correlation with gene and protein expression are unknown. To define host defense and immunosuppressive functions of normal density neutrophils (NDN) and LDN from COVID-19 patients, we recruited 64 patients with severe COVID-19 and 26 healthy donors (HD). Phagocytosis, respiratory burst activity, degranulation, neutrophil extracellular trap (NET) formation, and T-cell suppression in those neutrophil subsets were measured. NDN from severe/critical COVID-19 patients showed evidence of priming with enhanced phagocytosis, respiratory burst activity, and degranulation of secretory vesicles and gelatinase and specific granules, while NET formation was similar to HD NDN. COVID LDN response was impaired except for enhanced NET formation. A subset of COVID LDN with intermediate CD16 expression (CD16Int LDN) promoted T cell proliferation to a level similar to HD NDN, while COVID NDN and the CD16Hi LDN failed to stimulate T-cell activation. All 3 COVID-19 neutrophil populations suppressed stimulation of IFN-γ production, compared to HD NDN. We conclude that NDN and LDN from COVID-19 patients possess complementary functional capabilities that may act cooperatively to determine disease severity. We predict that global neutrophil responses that induce COVID-19 ARDS will vary depending on the proportion of neutrophil subsets.