RESUMEN
Vascular endothelial growth factor A (VEGF-A) is upregulated during hypoxia and is the major regulator of angiogenesis. VEGF-A expression has also been found to recruit myeloid cells to ischemic tissues where they contribute to angiogenesis. This study investigates the mechanisms underlying neutrophil recruitment to VEGF-A as well as the characteristics of these neutrophils. A previously undefined circulating subset of neutrophils shown to be CD49d(+)VEGFR1(high)CXCR4(high) was identified in mice and humans. By using chimeric mice with impaired VEGF receptor 1 (VEGFR1) or VEGFR2 signaling (Flt-1tk(-/-), tsad(-/-)), we found that parallel activation of VEGFR1 on neutrophils and VEGFR2 on endothelial cells was required for VEGF-A-induced recruitment of circulating neutrophils to tissue. Intravital microscopy of mouse microcirculation revealed that neutrophil recruitment by VEGF-A versus by the chemokine macrophage inflammatory protein 2 (MIP-2 [CXCL2]) involved the same steps of the recruitment cascade but that an additional neutrophil integrin (eg, VLA-4 [CD49d/CD29]) played a crucial role in neutrophil crawling and emigration to VEGF-A. Isolated CD49d(+) neutrophils featured increased chemokinesis but not chemotaxis compared with CD49d(-) neutrophils in the presence of VEGF-A. Finally, by targeting the integrin α4 subunit (CD49d) in a transplantation-based angiogenesis model that used avascular pancreatic islets transplanted to striated muscle, we demonstrated that inhibiting the recruitment of circulating proangiogenic neutrophils to hypoxic tissue impairs vessel neoformation. Thus, angiogenesis can be modulated by targeting cell-surface receptors specifically involved in VEGF-A-dependent recruitment of proangiogenic neutrophils without compromising recruitment of the neutrophil population involved in the immune response to pathogens.
Asunto(s)
Integrina alfa4/metabolismo , Islotes Pancreáticos/metabolismo , Músculo Esquelético/metabolismo , Neutrófilos/metabolismo , Receptores CXCR4/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/fisiología , Animales , Western Blotting , Células Cultivadas , Femenino , Citometría de Flujo , Humanos , Integrina alfa4/genética , Islotes Pancreáticos/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía por Video , Músculo Esquelético/citología , Neovascularización Fisiológica , Infiltración Neutrófila , Neutrófilos/citología , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores CXCR4/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
Lack of sleep greatly affects our immune system. The present study investigates the acute effects of total sleep deprivation on blood neutrophils, the most abundant immune cell in our circulation and the first cell type recruited to sites of infection. Thus, the population diversity and function of circulating neutrophils were compared in healthy young men following one night of total sleep deprivation (TSD) or after 8h regular sleep. We found that neutrophil counts were elevated after nocturnal wakefulness (2.0 ± 0.2 × 10(9)/l vs. 2.6 ± 0.2 × 10(9)/l, sleep vs. TSD, respectively) and the population contained more immature CD16(dim)/CD62L(bright) cells (0.11 ± 0.040 × 10(9)/l [5.5 ± 1.1%] vs. 0.26 ± 0.020 × 10(9)/l [9.9 ± 1.4%]). As the rise in numbers of circulating mature CD16(bright)/CD62L(bright) neutrophils was less pronounced, the fraction of this subpopulation showed a significant decrease (1.8 ± 0.15 × 10(9)/l [88 ± 1.8%] vs. 2.1 ± 0.12 × 10(9)/l [82 ± 2.8%]). The surface expression of receptors regulating mobilization of neutrophils from bone marrow was decreased (CXCR4 and CD49d on immature neutrophils; CXCR2 on mature neutrophils). The receptor CXCR2 is also involved in the production of reactive oxygen species (ROS), and in line with this, total neutrophils produced less ROS. In addition, following sleep loss, circulating neutrophils exhibited enhanced surface levels of CD11b, which indicates enhanced granular fusion and concomitant protein translocation to the membrane. Our findings demonstrate that sleep loss exerts significant effects on population diversity and function of circulating neutrophils in healthy men. To which extent these changes could explain as to why people with poor sleep patterns are more susceptible to infections warrants further investigation.
Asunto(s)
Neutrófilos/inmunología , Privación de Sueño/inmunología , Enfermedad Aguda , Antígeno CD11b/biosíntesis , Antígeno CD11b/genética , Núcleo Celular/ultraestructura , Quimiotaxis de Leucocito , Proteínas Ligadas a GPI/análisis , Voluntarios Sanos , Humanos , Selectina L/análisis , Recuento de Leucocitos , Masculino , Neutrófilos/química , Neutrófilos/clasificación , Neutrófilos/metabolismo , Polisomnografía , Especies Reactivas de Oxígeno/metabolismo , Receptores CXCR4/biosíntesis , Receptores CXCR4/genética , Receptores de IgG/análisis , Receptores de Interleucina-8B/biosíntesis , Receptores de Interleucina-8B/genética , Estallido Respiratorio , Adulto JovenRESUMEN
STUDY OBJECTIVES: To investigate whether total sleep deprivation (TSD) affects circulating concentrations of neuron-specific enolase (NSE) and S100 calcium binding protein B (S-100B) in humans. These factors are usually found in the cytoplasm of neurons and glia cells. Increasing concentrations of these factors in blood may be therefore indicative for either neuronal damage, impaired blood brain barrier function, or both. In addition, amyloid ß (Aß) peptides 1-42 and 1-40 were measured in plasma to calculate their ratio. A reduced plasma ratio of Aß peptides 1-42 to 1-40 is considered an indirect measure of increased deposition of Aß 1-42 peptide in the brain. DESIGN: Subjects participated in two conditions (including either 8-h of nocturnal sleep [22:30-06:30] or TSD). Fasting blood samples were drawn before and after sleep interventions (19:30 and 07:30, respectively). SETTING: Sleep laboratory. PARTICIPANTS: 15 healthy young men. RESULTS: TSD increased morning serum levels of NSE (P = 0.002) and S-100B (P = 0.02) by approximately 20%, compared with values obtained after a night of sleep. In contrast, the ratio of Aß peptides 1-42 to 1-40 did not differ between the sleep interventions. CONCLUSIONS: Future studies in which both serum and cerebrospinal fluid are sampled after sleep loss should elucidate whether the increase in serum neuron-specific enolase and S100 calcium binding protein B is primarily caused by neuronal damage, impaired blood brain barrier function, or is just a consequence of increased gene expression in non-neuronal cells, such as leukocytes.
Asunto(s)
Fosfopiruvato Hidratasa/sangre , Proteínas S100/sangre , Privación de Sueño/sangre , Enfermedad Aguda , Péptidos beta-Amiloides/sangre , Ayuno/sangre , Voluntarios Sanos , Humanos , Masculino , Fragmentos de Péptidos/sangre , Sueño/fisiología , Factores de Tiempo , Adulto JovenRESUMEN
αB-crystallin is a small heat shock protein, which has pro-angiogenic properties by increasing survival of endothelial cells and secretion of vascular endothelial growth factor A. Here we demonstrate an additional role of αB-crystallin in regulating vascular function, through enhancing tumor necrosis factor α (TNF-α) induced expression of endothelial adhesion molecules involved in leukocyte recruitment. Ectopic expression of αB-crystallin in endothelial cells increases the level of E-selectin expression in response to TNF-α, and enhances leukocyte-endothelial interaction in vitro. Conversely, TNF-α-induced expression of intercellular adhesion molecule 1, vascular cell adhesion molecule 1 and E-selectin is markedly inhibited in endothelial cells isolated from αB-crystallin-deficient mice. This is associated with elevated levels of IκB in αB-crystallin deficient cells and incomplete degradation upon TNF-α stimulation. Consistent with this, endothelial adhesion molecule expression is reduced in inflamed vessels of αB-crystallin deficient mice, and leukocyte rolling velocity is increased. Our data identify αB-crystallin as a new regulator of leukocyte recruitment, by enhancing pro-inflammatory nuclear factor κ B-signaling and endothelial adhesion molecule expression during endothelial activation.
Asunto(s)
Moléculas de Adhesión Celular/biosíntesis , Células Endoteliales/citología , Rodamiento de Leucocito/fisiología , Leucocitos/citología , FN-kappa B/metabolismo , Cadena B de alfa-Cristalina/fisiología , alfa-Cristalinas/deficiencia , Transporte Activo de Núcleo Celular , Animales , Adhesión Celular/fisiología , Moléculas de Adhesión Celular/genética , Línea Celular , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana , Humanos , Proteínas I-kappa B/biosíntesis , Proteínas I-kappa B/genética , Inflamación , Células Jurkat , Masculino , Ratones , Microvasos/citología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción Genética , Factor de Necrosis Tumoral alfa/fisiología , Regulación hacia Arriba , Cadena B de alfa-Cristalina/genética , alfa-Cristalinas/genéticaRESUMEN
Recruitment and retention of leukocytes at a site of blood vessel growth are crucial for proper angiogenesis and subsequent tissue perfusion. Although critical for many aspects of regenerative medicine, the mechanisms of leukocyte recruitment to and actions at sites of angiogenesis are not fully understood. In this study, we investigated the signals attracting leukocytes to avascular transplanted pancreatic islets and leukocyte actions at the engraftment site. Expression of the angiogenic stimulus VEGF-A by mouse pancreatic islets was elevated shortly after syngeneic transplantation to muscle. High levels of leukocytes, predominantly CD11b(+)/Gr-1(+)/CXCR4(hi) neutrophils, were observed at the site of engraftment, whereas VEGF-A-deficient islets recruited only half of the amount of leukocytes when transplanted. Acute VEGF-A exposure of muscle increased leukocyte extravasation but not the levels of SDF-1α. VEGF-A-recruited neutrophils expressed 10 times higher amounts of MMP-9 than neutrophils recruited to an inflammatory stimulus. Revascularization of islets transplanted to MMP-9-deficient mice was impaired because blood vessels initially failed to penetrate grafts, and after 2 weeks vascularity was still disturbed. This study demonstrates that VEGF-A recruits a proangiogenic circulating subset of CD11b(+)/Gr-1(+) neutrophils that are CXCR4(hi) and deliver large amounts of the effector protein MMP-9, required for islet revascularization and functional integration after transplantation.
Asunto(s)
Trasplante de Islotes Pancreáticos/fisiología , Metaloproteinasa 9 de la Matriz/metabolismo , Neovascularización Fisiológica/fisiología , Neutrófilos/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Antígeno CD11b/metabolismo , Quimiocina CXCL12/metabolismo , Femenino , Hipoxia , Inmunohistoquímica , Islotes Pancreáticos/irrigación sanguínea , Islotes Pancreáticos/metabolismo , Masculino , Metaloproteinasa 9 de la Matriz/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Microscopía por Video , Neovascularización Fisiológica/genética , Infiltración Neutrófila , Receptores CXCR4 , Receptores de Quimiocina/metabolismo , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
Regulation of vascular endothelial (VE) growth factor (VEGF)-induced permeability is critical in physiological and pathological processes. We show that tyrosine phosphorylation of VEGF receptor 2 (VEGFR2) at Y951 facilitates binding of VEGFR2 to the Rous sarcoma (Src) homology 2-domain of T cell-specific adaptor (TSAd), which in turn regulates VEGF-induced activation of the c-Src tyrosine kinase and vascular permeability. c-Src was activated in vivo and in vitro in a VEGF/TSAd-dependent manner, and was regulated via increased phosphorylation at pY418 and reduced phosphorylation at pY527. Tsad silencing blocked VEGF-induced c-Src activation, but did not affect pathways involving phospholipase Cγ, extracellular regulated kinase, and endothelial nitric oxide. VEGF-induced rearrangement of VE-cadherin-positive junctions in endothelial cells isolated from mouse lungs, or in mouse cremaster vessels, was dependent on TSAd expression, and TSAd formed a complex with VE-cadherin, VEGFR2, and c-Src at endothelial junctions. Vessels in tsad(-/-) mice showed undisturbed flow and pressure, but impaired VEGF-induced permeability, as measured by extravasation of Evans blue, dextran, and microspheres in the skin and the trachea. Histamine-induced extravasation was not affected by TSAd deficiency. We conclude that TSAd is required for VEGF-induced, c-Src-mediated regulation of endothelial cell junctions and for vascular permeability.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Permeabilidad Capilar/fisiología , Transducción de Señal/fisiología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Familia-src Quinasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Antígenos CD/metabolismo , Western Blotting , Cadherinas/metabolismo , Permeabilidad Capilar/efectos de los fármacos , Células Cultivadas , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Extravasación de Materiales Terapéuticos y Diagnósticos/etiología , Femenino , Fluoresceína-5-Isotiocianato/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Microesferas , Fosforilación/efectos de los fármacos , Unión Proteica , Interferencia de ARN , Transducción de Señal/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/farmacologíaRESUMEN
Nitric oxide (NO) generated by vascular NO synthases can exert anti-inflammatory effects, partly through its ability to decrease leukocyte recruitment. Inorganic nitrate and nitrite, from endogenous or dietary sources, have emerged as alternative substrates for NO formation in mammals. Bioactivation of nitrate is believed to require initial reduction to nitrite by oral commensal bacteria. Here we investigated the effects of inorganic nitrate and nitrite on leukocyte recruitment in microvascular inflammation and in NSAID-induced small-intestinal injury. We show that leukocyte emigration in response to the proinflammatory chemokine MIP-2 is reduced by 70% after 7 days of dietary nitrate supplementation as well as by acute intravenous nitrite administration. Nitrite also reduced leukocyte adhesion to a similar extent and this effect was inhibited by the soluble guanylyl cyclase inhibitor ODQ, whereas the effect on emigrated leukocytes was not altered by this treatment. Further studies in TNF-α-stimulated endothelial cells revealed that nitrite dose-dependently reduced the expression of ICAM-1. In rats and mice subjected to a challenge with diclofenac, dietary nitrate prevented the increase in myeloperoxidase and P-selectin levels in small-intestinal tissue. Antiseptic mouthwash, which eliminates oral nitrate reduction, markedly blunted the protective effect of dietary nitrate on P-selectin levels. Despite attenuation of the acute immune response, the overall ability to clear an infection with Staphylococcus aureus was not suppressed by dietary nitrate as revealed by noninvasive IVIS imaging. We conclude that dietary nitrate markedly reduces leukocyte recruitment to inflammation in a process involving attenuation of P-selectin and ICAM-1 upregulation. Bioactivation of dietary nitrate requires intermediate formation of nitrite by oral nitrate-reducing bacteria and then probably further reduction to NO and other bioactive nitrogen oxides in the tissues.
Asunto(s)
Intestino Delgado/patología , Microvasos/patología , Infiltración Neutrófila/efectos de los fármacos , Nitratos/farmacología , Nitritos/farmacología , Animales , Antiinflamatorios no Esteroideos/efectos adversos , Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Quimiocina CXCL2 , GMP Cíclico/metabolismo , Diclofenaco/efectos adversos , Suplementos Dietéticos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Expresión Génica/efectos de los fármacos , Humanos , Inflamación/inducido químicamente , Inflamación/tratamiento farmacológico , Inflamación/patología , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , Intestino Delgado/irrigación sanguínea , Intestino Delgado/inmunología , Intestino Delgado/metabolismo , Recuento de Leucocitos , Masculino , Ratones , Ratones Endogámicos C57BL , Microvasos/efectos de los fármacos , Antisépticos Bucales/farmacología , Nitratos/administración & dosificación , Nitratos/uso terapéutico , Nitritos/administración & dosificación , Nitritos/uso terapéutico , Selectina-P/genética , Selectina-P/metabolismo , Peroxidasa/genética , Peroxidasa/metabolismo , Ratas , Ratas Sprague-Dawley , Infecciones Estafilocócicas/tratamiento farmacológico , Staphylococcus aureus/efectos de los fármacosRESUMEN
BACKGROUND: Patients suffering from diabetes show defective bacterial clearance. This study investigates the effects of elevated plasma glucose levels during diabetes on leukocyte recruitment and function in established models of inflammation. METHODOLOGY/PRINCIPAL FINDINGS: Diabetes was induced in C57Bl/6 mice by intravenous alloxan (causing severe hyperglycemia), or by high fat diet (moderate hyperglycemia). Leukocyte recruitment was studied in anaesthetized mice using intravital microscopy of exposed cremaster muscles, where numbers of rolling, adherent and emigrated leukocytes were quantified before and during exposure to the inflammatory chemokine MIP-2 (0.5 nM). During basal conditions, prior to addition of chemokine, the adherent and emigrated leukocytes were increased in both alloxan- (62±18% and 85±21%, respectively) and high fat diet-induced (77±25% and 86±17%, respectively) diabetes compared to control mice. MIP-2 induced leukocyte emigration in all groups, albeit significantly more cells emigrated in alloxan-treated mice (15.3±1.0) compared to control (8.0±1.1) mice. Bacterial clearance was followed for 10 days after subcutaneous injection of bioluminescent S. aureus using non-invasive IVIS imaging, and the inflammatory response was assessed by Myeloperoxidase-ELISA and confocal imaging. The phagocytic ability of leukocytes was assessed using LPS-coated fluorescent beads and flow cytometry. Despite efficient leukocyte recruitment, alloxan-treated mice demonstrated an impaired ability to clear bacterial infection, which we found correlated to a 50% decreased phagocytic ability of leukocytes in diabetic mice. CONCLUSIONS/SIGNIFICANCE: These results indicate that reduced ability to clear bacterial infections observed during experimentally induced diabetes is not due to reduced leukocyte recruitment since sustained hyperglycemia results in increased levels of adherent and emigrated leukocytes in mouse models of type 1 and type 2 diabetes. Instead, decreased phagocytic ability observed for leukocytes isolated from diabetic mice might account for the impaired bacterial clearance.
Asunto(s)
Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 2/inmunología , Leucocitos/inmunología , Animales , Glucemia/metabolismo , Adhesión Celular/efectos de los fármacos , Recuento de Células , Movimiento Celular/efectos de los fármacos , Quimiocina CXCL2/farmacología , Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 1/complicaciones , Diabetes Mellitus Tipo 1/microbiología , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/microbiología , Dieta Alta en Grasa/efectos adversos , Modelos Animales de Enfermedad , Hiperglucemia/inducido químicamente , Hiperglucemia/complicaciones , Inflamación/sangre , Inflamación/complicaciones , Inflamación/inmunología , Inflamación/microbiología , Leucocitos/citología , Leucocitos/efectos de los fármacos , Leucocitos/microbiología , Masculino , Ratones , Ratones Endogámicos C57BL , Fagocitos/citología , Fagocitos/efectos de los fármacos , Fagocitos/microbiología , Staphylococcus aureus/fisiologíaRESUMEN
During infection, chemokines sequestered on endothelium induce recruitment of circulating leukocytes into the tissue where they chemotax along chemokine gradients toward the afflicted site. The aim of this in vivo study was to determine whether a chemokine gradient was formed intravascularly and influenced intraluminal neutrophil crawling and transmigration. A chemokine gradient was induced by placing a macrophage inflammatory protein-2 (MIP-2)-containing (CXCL2) gel on the cremaster muscle of anesthetized wild-type mice or heparanase-overexpressing transgenic mice (hpa-tg) with truncated heparan sulfate (HS) side chains. Neutrophil-endothelial interactions were visualized by intravital microscopy and chemokine gradients detected by confocal microscopy. Localized extravascular chemokine release (MIP-2 gel) induced directed neutrophil crawling along a chemotactic gradient immobilized on the endothelium and accelerated their recruitment into the target tissue compared with homogeneous extravascular chemokine concentration (MIP-2 superfusion). Endothelial chemokine sequestration occurred exclusively in venules and was HS-dependent, and neutrophils in hpa-tg mice exhibited random crawling. Despite similar numbers of adherent neutrophils in hpa-tg and wild-type mice, the altered crawling in hpa-tg mice was translated into decreased number of emigrated neutrophils and ultimately decreased the ability to clear bacterial infections. In conclusion, an intravascular chemokine gradient sequestered by endothelial HS effectively directs crawling leukocytes toward transmigration loci close to the infection site.