RESUMEN
The regulation of actin dynamics is pivotal for cellular processes such as cell adhesion, migration, and phagocytosis and thus is crucial for neutrophils to fulfill their roles in innate immunity. Many factors have been implicated in signal-induced actin polymerization, but the essential nature of the potential negative modulators are still poorly understood. Here we report that NADPH oxidase-dependent physiologically generated reactive oxygen species (ROS) negatively regulate actin polymerization in stimulated neutrophils via driving reversible actin glutathionylation. Disruption of glutaredoxin 1 (Grx1), an enzyme that catalyzes actin deglutathionylation, increased actin glutathionylation, attenuated actin polymerization, and consequently impaired neutrophil polarization, chemotaxis, adhesion, and phagocytosis. Consistently, Grx1-deficient murine neutrophils showed impaired in vivo recruitment to sites of inflammation and reduced bactericidal capability. Together, these results present a physiological role for glutaredoxin and ROS- induced reversible actin glutathionylation in regulation of actin dynamics in neutrophils.
Asunto(s)
Actinas/metabolismo , Neutrófilos/inmunología , Neutrófilos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Infecciones Bacterianas/genética , Infecciones Bacterianas/inmunología , Células Cultivadas , Quimiotaxis/inmunología , Glutarredoxinas/genética , Glutarredoxinas/inmunología , Humanos , Ratones , Ratones Noqueados , NADPH Oxidasas/metabolismo , Unión Proteica , Seudópodos/metabolismoRESUMEN
BACKGROUND: Allergen immunotherapy is currently the only disease-modifying treatment available for allergic rhinitis and allergic asthma. OBJECTIVES: We sought to evaluate the induction of sustained tolerance to allergen when anti-IL-4 was combined with a suboptimal course of grass pollen subcutaneous immunotherapy (SCIT) using the allergen-induced skin late-phase response (LPR) and exploratory immune monitoring as surrogate markers of therapeutic response. METHODS: In this randomized, double-blind, 3-group parallel design trial, 37 participants with seasonal allergic rhinitis received suboptimal SCIT (30,000 standardized quality units) in combination with anti-IL-4 (VAK694) and suboptimal SCIT (30,000 standardized quality units) plus placebo antibody or double placebo (placebo SCIT and placebo antibody) restricted to 13 weeks before the grass pollen season. The primary end point was the size of the LPR at 12 months. Exploratory end points included measures of the immunomodulatory activity of treatment by using IL-4 and IL-10 FluoroSpot assays, flow cytometry of T cells, and measurement of IgE, IgG4, and facilitated antigen binding. RESULTS: Both active treatment arms led to a substantial and sustained reduction of the LPR with no additional suppression with addition of anti-IL-4. Treatment with anti-IL-4 and SCIT compared with SCIT alone led to a sustained reduction in allergen-specific IL-4-producing cell counts (P < .01). Both active treatment arms led to induction of dual IL-4/IL-10-producing cells during the pollen season. CONCLUSION: The combination of anti-IL-4 with SCIT provided no additional benefit over SCIT alone in suppressing the allergen-induced skin LPR. A larger trial is needed to assess whether the observed ex vivo downregulation of TH2 responses might translate into clinical benefit.
Asunto(s)
Alérgenos/inmunología , Anticuerpos Monoclonales/uso terapéutico , Desensibilización Inmunológica , Poaceae/efectos adversos , Polen/inmunología , Rinitis Alérgica Estacional/inmunología , Rinitis Alérgica Estacional/terapia , Adulto , Alérgenos/administración & dosificación , Anticuerpos Monoclonales/farmacología , Biomarcadores , Terapia Combinada , Desensibilización Inmunológica/efectos adversos , Desensibilización Inmunológica/métodos , Femenino , Humanos , Inmunoglobulina E/sangre , Inmunoglobulina E/inmunología , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Inyecciones Subcutáneas , Interleucina-10/metabolismo , Interleucina-4/antagonistas & inhibidores , Masculino , Persona de Mediana Edad , Rinitis Alérgica Estacional/diagnóstico , Rinitis Alérgica Estacional/tratamiento farmacológico , Factores de Riesgo , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Resultado del Tratamiento , Adulto JovenRESUMEN
Neutrophil chemotaxis plays an essential role in innate immunity, but the underlying cellular mechanism is still not fully characterized. Here, using a small-molecule functional screening, we identified NADPH oxidase-dependent reactive oxygen species as key regulators of neutrophil chemotactic migration. Neutrophils with pharmacologically inhibited oxidase, or isolated from chronic granulomatous disease (CGD) patients and mice, formed more frequent multiple pseudopodia and lost their directionality as they migrated up a chemoattractant concentration gradient. Knocking down NADPH oxidase in differentiated neutrophil-like HL60 cells also led to defective chemotaxis. Consistent with the in vitro results, adoptively transferred CGD murine neutrophils showed impaired in vivo recruitment to sites of inflammation. Together, these results present a physiological role for reactive oxygen species in regulating neutrophil functions and shed light on the pathogenesis of CGD.
Asunto(s)
Quimiotaxis , Enfermedad Granulomatosa Crónica/inmunología , Neutrófilos/inmunología , Especies Reactivas de Oxígeno/metabolismo , Bibliotecas de Moléculas Pequeñas , Animales , Evaluación Preclínica de Medicamentos , Técnicas de Silenciamiento del Gen , Enfermedad Granulomatosa Crónica/enzimología , Células HL-60 , Humanos , Ratones , Ratones Endogámicos , NADPH Oxidasas/antagonistas & inhibidores , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Neutrófilos/efectos de los fármacos , Neutrófilos/enzimologíaRESUMEN
We generated Fas-activated serine threonine phosphoprotein (FAST)-deficient mice (FAST(-/-)) to study the in vivo role of FAST in immune system function. In a model of house dust mite-induced allergic pulmonary inflammation, wild type mice develop a mixed cellular infiltrate composed of eosinophils, lymphocytes, and neutrophils. FAST(-/-) mice develop airway inflammation that is distinguished by the near absence of neutrophils. Similarly, LPS-induced alveolar neutrophil recruitment is markedly reduced in FAST(-/-) mice compared with wild type controls. This is accompanied by reduced concentrations of cytokines (TNF-alpha and IL-6 and -23) and chemoattractants (MIP-2 and keratinocyte chemoattractant) in bronchoalveolar lavage fluids. Because FAST(-/-) neutrophils exhibit normal chemotaxis and survival, impaired neutrophil recruitment is likely to be due to reduced production of chemoattractants within the pulmonary parenchyma. Studies using bone marrow chimeras implicate lung resident hematopoietic cells (e.g., pulmonary dendritic cells and/or alveolar macrophages) in this process. In conclusion, our results introduce FAST as a proinflammatory factor that modulates the function of lung resident hematopoietic cells to promote neutrophil recruitment and pulmonary inflammation.
Asunto(s)
Proteínas Mitocondriales/fisiología , Fosfoproteínas/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Hipersensibilidad Respiratoria/inmunología , Hipersensibilidad Respiratoria/patología , Receptor fas/fisiología , Lesión Pulmonar Aguda/genética , Lesión Pulmonar Aguda/inmunología , Lesión Pulmonar Aguda/patología , Alérgenos/administración & dosificación , Alérgenos/inmunología , Animales , Células Cultivadas , Quimiotaxis de Leucocito/genética , Quimiotaxis de Leucocito/inmunología , Dermatophagoides pteronyssinus/inmunología , Polvo/inmunología , Femenino , Hematopoyesis/genética , Hematopoyesis/inmunología , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Mutantes , Ratones Transgénicos , Proteínas Mitocondriales/deficiencia , Proteínas Mitocondriales/genética , Infiltración Neutrófila/genética , Infiltración Neutrófila/inmunología , Fosfoproteínas/deficiencia , Fosfoproteínas/genética , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/genética , Proteínas de Unión al ARN/fisiología , Hipersensibilidad Respiratoria/genética , Antígeno Intracelular 1 de las Células TRESUMEN
Various neutrophil functions such as phagocytosis, superoxide production, and survival are regulated by integrin signaling. Despite the essential role of focal adhesion kinase (FAK) in mediating this signaling pathway, its exact function in neutrophils is ill defined. In this study, we investigated the role of FAK in neutrophils using a myeloid-specific conditional FAK knockout mouse. As reported in many other cell types, FAK is required for regulation of focal adhesion dynamics when neutrophils adhere to fibronectin or ICAM-1. Adhesion on VCAM-1-coated surfaces and chemotaxis after adhesion were not altered in FAK null neutrophils. In addition, we observed significant reduction in NADPH oxidase-mediated superoxide production and complement-mediated phagocytosis in FAK null neutrophils. As a result, these neutrophils displayed decreased pathogen killing capability both in vitro and in vivo in a mouse peritonitis model. In adherent cells, the defects associated with FAK deficiency are likely due to suppression of phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) signaling and chemoattractant-elicited calcium signaling. Disruption of FAK also reduced chemoattractant-elicited superoxide production in suspended neutrophils in the absence of cell adhesion. This may be solely caused by suppression of PtdIns(3,4,5)P3 signaling in these cells, because the fMLP-elicited calcium signal was not altered. Consistent with decreased PtdIns(3,4,5)P3/Akt signaling in FAK null neutrophils, we also observed accelerated spontaneous death in these cells. Taken together, our results revealed previously unrecognized roles of FAK in neutrophil function and provided a potential therapeutic target for treatment of a variety of infectious and inflammatory diseases.
Asunto(s)
Adhesión Celular/inmunología , Proteína-Tirosina Quinasas de Adhesión Focal/fisiología , Neutrófilos/inmunología , Fagocitosis , Transducción de Señal/inmunología , Animales , Señalización del Calcio , Moléculas de Adhesión Celular/metabolismo , Muerte Celular , Quimiotaxis , Proteína-Tirosina Quinasas de Adhesión Focal/deficiencia , Ratones , Ratones Noqueados , Neutrófilos/citología , Peritonitis/inmunología , Fosfatos de Fosfatidilinositol/metabolismoRESUMEN
Protein drugs that neutralize vascular endothelial growth factor (VEGF), such as aflibercept or ranibizumab, rescue vision in patients with retinal vascular diseases. Nonetheless, optimal visual outcomes require intraocular injections as frequently as every month. Here we report a method to extend the intravitreal half-life of protein drugs as an alternative to either encapsulation or chemical modifications with polymers. We combine a 97-amino-acid peptide of human origin that binds hyaluronan, a major macromolecular component of the eye's vitreous, with therapeutic antibodies and proteins. When administered to rabbit and monkey eyes, the half-life of the modified proteins is increased â¼3-4-fold relative to unmodified proteins. We further show that prototype long-acting anti-VEGF drugs (LAVAs) that include this peptide attenuate VEGF-induced retinal changes in animal models of neovascular retinal disease â¼3-4-fold longer than unmodified drugs. This approach has the potential to reduce the dosing frequency associated with retinal disease treatments.
Asunto(s)
Bevacizumab/administración & dosificación , Ranibizumab/administración & dosificación , Receptores de Factores de Crecimiento Endotelial Vascular/administración & dosificación , Proteínas Recombinantes de Fusión/administración & dosificación , Enfermedades de la Retina/tratamiento farmacológico , Inhibidores de la Angiogénesis/administración & dosificación , Inhibidores de la Angiogénesis/química , Inhibidores de la Angiogénesis/farmacocinética , Animales , Bevacizumab/química , Bevacizumab/farmacocinética , Modelos Animales de Enfermedad , Femenino , Semivida , Humanos , Ácido Hialurónico/química , Inyecciones Intravítreas , Macaca fascicularis , Masculino , Conejos , Ranibizumab/química , Ranibizumab/farmacocinética , Receptores de Factores de Crecimiento Endotelial Vascular/química , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/farmacocinética , Enfermedades de la Retina/metabolismoRESUMEN
The second messenger phosphatidylinositol(3,4,5)P(3) (PtdIns(3,4,5)P(3)) is formed by stimulation of various receptors, including G protein-coupled receptors and integrins. The lipid phosphatases PTEN and SHIP1 are critical in regulating the level of PtdIns(3,4,5)P(3) during chemotaxis. Observations that loss of PTEN had minor and loss of SHIP1 resulted in a severe chemotaxis defect in neutrophils led to the belief that SHIP1 rather than PTEN acts as a predominant phospholipid phosphatase in establishing a PtdIns(3,4,5)P(3) compass. In this study, we show that SHIP1 regulates PtdIns(3,4,5)P(3) production in response to cell adhesion and plays a limited role when cells are in suspension. SHIP1((-)/(-)) neutrophils lose their polarity upon cell adhesion and are extremely adherent, which impairs chemotaxis. However, chemo-taxis can be restored by reducing adhesion. Loss of SHIP1 elevates Akt activation following cell adhesion due to increased PtdIns(3,4,5)P(3) production. From our observations, we conclude that SHIP1 prevents formation of top-down PtdIns(3,4,5)P(3) polarity to facilitate proper cell attachment and detachment during chemotaxis.
Asunto(s)
Adhesión Celular/fisiología , Movimiento Celular/fisiología , Fosfohidrolasa PTEN/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Animales , Polaridad Celular/fisiología , Quimiotaxis de Leucocito/fisiología , Técnicas In Vitro , Inositol Polifosfato 5-Fosfatasas , Ratones , Ratones Noqueados , Modelos Biológicos , Neutrófilos/fisiología , Fosfohidrolasa PTEN/deficiencia , Fosfohidrolasa PTEN/genética , Fosfatos de Fosfatidilinositol/biosíntesis , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas , Monoéster Fosfórico Hidrolasas/química , Monoéster Fosfórico Hidrolasas/deficiencia , Monoéster Fosfórico Hidrolasas/genética , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Sistemas de Mensajero Secundario , Tirosina/químicaRESUMEN
Neutrophil chemotaxis is a critical component in innate immunity. Recently, using a small-molecule functional screening, we identified NADPHoxidase- dependent Reactive Oxygen Species (ROS) as key regulators of neutrophil chemotactic migration. Neutrophils depleted of ROS form more frequent multiple pseudopodia and lost their directionality as they migrate up a chemoattractant concentration gradient. Here, we further studied the role of ROS in neutrophil chemotaxis and found that multiple pseudopodia formation induced by NADPH inhibitor diphenyleneiodonium chloride (DPI) was more prominent in relatively shallow chemoattractant gradient. It was reported that, in shallow chemoattractant gradients, new pseudopods are usually generated when existing ones bifurcate. Directional sensing is mediated by maintaining the most accurate existing pseudopod, and destroying pseudopods facing the wrong direction by actin depolymerization. We propose that NADPH-mediated ROS production may be critical for disruption of misoriented pseudopods in chemotaxing neutrophils. Thus, inhibition of ROS production will lead to formation of multiple pseudopodia.
RESUMEN
Cancer cells in their respective microenvironments must endure various growth-constraining stresses. Under these conditions, the cancer cell-derived factors are thought to modulate the signaling pathways between cell growth and dormancy. Here, we describe a cancer cell-derived regulatory system that modulates the phosphatidylinositol 3'-kinase (PI3K)-Akt pathway under serum deprivation stress. Through the use of biochemical purification, we reveal that cancer cell-secreted insulin-like growth factor 1 (IGF-1) and clusterin, an extracellular stress protein, constitute this regulatory system. We show that secreted clusterin associates with IGF-1 and inhibits its binding to the IGF-1 receptor and hence negatively regulates the PI3K-Akt pathway during serum deprivation. This inhibitory function of clusterin appears to prefer IGF-1, as it fails to exert any effects on epidermal growth factor signaling. We demonstrate furthermore that the constitutive activation of oncogenic signaling downstream of IGF-1 confers insensitivity to the inhibitory effects of clusterin. Thus, the interplay between cancer cell-derived clusterin and IGF-1 may dictate the outcome of cell growth and dormancy during tumorigenic progression.
Asunto(s)
Clusterina/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Neoplasias/enzimología , Neoplasias/patología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Proliferación Celular/efectos de los fármacos , Clusterina/química , Clusterina/aislamiento & purificación , Medio de Cultivo Libre de Suero , Activación Enzimática/efectos de los fármacos , Factor de Crecimiento Epidérmico/farmacología , Humanos , Ratones , Datos de Secuencia Molecular , Unión Proteica/efectos de los fármacos , Receptor IGF Tipo 1/metabolismo , Transducción de Señal/efectos de los fármacos , Pez CebraRESUMEN
All-trans retinoic acid (ATRA) has been widely used in differentiation therapy for acute promyelocytic leukemia (APL). ATRA binds to retinoic acid receptor (RAR) and triggers the formation of the transcription coactivator complex, which leads to changes in gene expression, APL cell-cycle arrest and differentiation, and clinical remission. The mechanisms responsible for ATRA's beneficial effects are still ill-defined. Here, we conducted a large-scale, unbiased short hairpin RNA (shRNA) screen aiming to identify mediators of ATRA-induced differentiation and growth arrest of APL cells. Twenty-six proteins were identified. They cover a wide range of cellular functions, including gene expression, intracellular signaling, cell death control, stress responses, and metabolic regulation, indicating the complexity of ATRA-induced cell growth control and differentiation in APL. One of these proteins, the ubiquitin-conjugating enzyme UBE2D3, is up-regulated in ATRA-treated acute promyelocytic NB4 cells. UBE2D3 is physically associated with cyclin D1 and mediates ATRA-induced cyclin D1 degradation. Knocking down UBE2D3 by RNA interference (RNAi) leads to blockage of ATRA-induced cyclin D1 degradation and cell-cycle arrest. Thus, our results highlight the involvement of the ubiquitin-mediated proteolysis pathway in ATRA-induced cell-cycle arrest and provide a novel strategy for modulating ATRA-elicited cellular effects.
Asunto(s)
División Celular/efectos de los fármacos , Leucemia Promielocítica Aguda/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , Tretinoina/farmacología , Enzimas Ubiquitina-Conjugadoras/genética , Ciclo Celular , Línea Celular Tumoral , Resistencia a Antineoplásicos , Humanos , Leucemia Promielocítica Aguda/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Transducción de SeñalRESUMEN
The recruitment and activation of neutrophils at infected tissues is essential for host defense against invading microorganisms. However, excessive neutrophil recruitment or activation can also damage the surrounding tissues and cause unwanted inflammation. Hence, the responsiveness of neutrophils needs to be tightly regulated. In this study, we have investigated the functional role of tumor suppressor PTEN in neutrophils by using a mouse line in which PTEN is disrupted only in myeloid-derived cells. Chemoattractant-stimulated PTEN(-/-) neutrophils displayed significantly higher Akt phosphorylation and actin polymerization. A larger fraction of these neutrophils displayed membrane ruffles in response to chemoattractant stimulation. In addition, chemoattractant-induced transwell migration and superoxide production were also augmented. Single-cell chemotaxis assays showed that PTEN(-/-) neutrophils have a small (yet statistically significant) defect in directionality. However, these neutrophils also showed an increase in cell speed. As a result, overall chemotaxis, which depends on speed and directionality, was not affected. Consistent with the increased responsiveness of PTEN(-/-) neutrophils, the in vivo recruitment of these cells to the inflamed peritoneal cavity was significantly enhanced. Thus, as a physiologic-negative regulator, PTEN should be a promising therapeutic target for modulating neutrophil functions in various infectious and inflammatory diseases.
Asunto(s)
Quimiotaxis de Leucocito/fisiología , Infiltración Neutrófila/fisiología , Neutrófilos/metabolismo , Fosfohidrolasa PTEN/metabolismo , Animales , Línea Celular , Factores Quimiotácticos/farmacología , Quimiotaxis de Leucocito/efectos de los fármacos , Inflamación/genética , Inflamación/metabolismo , Ratones , Ratones Noqueados , Infiltración Neutrófila/efectos de los fármacos , Neutrófilos/citología , Fosfohidrolasa PTEN/deficiencia , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
Many neutrophil functions are regulated by phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3) that mediates protein membrane translocation via binding to pleckstrin homolog (PH) domains within target proteins. Here we show that inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4), a cytosolic small molecule, bound the same PH domain of target proteins and competed for binding to PtdIns(3,4,5)P3. In neutrophils, chemoattractant stimulation triggered rapid elevation in Ins(1,3,4,5)P4 concentration. Depletion of Ins(1,3,4,5)P4 by deleting the gene encoding InsP3KB, which converts Ins(1,4,5)P3 to Ins(1,3,4,5)P4, enhanced membrane translocation of the PtdIns(3,4,5)P3-specific PH domain. This led to enhanced sensitivity to chemoattractant stimulation, elevated superoxide production, and enhanced neutrophil recruitment to inflamed peritoneal cavity. On the contrary, augmentation of intracellular Ins(1,3,4,5)P4 concentration blocked PH domain-mediated membrane translocation of target proteins and dramatically decreased the sensitivity of neutrophils to chemoattractant stimulation. These findings establish a role for Ins(1,3,4,5)P4 in cellular signal transduction pathways and provide another mechanism for modulating PtdIns(3,4,5)P3 signaling in neutrophils.
Asunto(s)
Fosfatos de Inositol/metabolismo , Neutrófilos/inmunología , Neutrófilos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Transducción de Señal/inmunología , Animales , Cromatografía Líquida de Alta Presión , Humanos , Inmunoprecipitación , Ratones , Transporte de Proteínas/inmunologíaRESUMEN
Neutrophil spontaneous death plays essential roles in neutrophil homeostasis and resolution of inflammation, whereas the underlying molecular mechanisms are still ill-defined. Neutrophils die because of programmed cell death or apoptosis. However, treatment with inhibitor of caspases, which are responsible for the majority of apoptotic cell deaths, does not prevent the spontaneous death of neutrophils. PKB/Akt possesses prosurvival and antiapoptotic activities in a variety of cells. In this study, we show that Akt activity decreases dramatically during the course of neutrophil death. Both phosphatidylinositol 3-kinase and Akt inhibitors enhance neutrophil death. Conditions delaying neutrophil death, such as treatment with granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor, or IFN-gamma, restore Akt activity. Finally, we demonstrate that neutrophils depleted of PTEN, a phosphatidylinositol 3'-phosphatase that negatively regulates Akt activity, live much longer than WT neutrophils. Thus, we establish Akt deactivation as a causal mediator of neutrophil spontaneous death.