RESUMEN
In a lipopolysaccharide (LPS)-induced rat model of sepsis (endotoxaemia), we previously demonstrated that pravastatin reduced microvascular inflammation via increased endothelial nitric oxide synthase III (NOSIII). This study aimed to determine whether atorvastatin, the most commonly used statin for lowering cholesterol, exerted beneficial pleiotropic effects via a similar mechanism. The mesenteric microcirculation of anaesthetised male Wistar rats (308 ± 63 g, n = 54) was prepared for fluorescent intravital microscopy. Over 4 h, animals received intravenous (i.v.) administration of either saline, LPS (150 µg kg(-1) h(-1)) or LPS + atorvastatin (200 µg kg(-1) s.c., 18 and 3 h before LPS), with/without the non-specific NOS inhibitor L-NG-Nitroarginine Methyl Ester (L-NAME) (10 µg kg(-1) h(-1)) or NOSII-specific inhibitor 1400 W (20 µg kg(-1) min(-1)). LPS decreased mean arterial blood pressure (MAP) (4 h, control 113 ± 20 mmHg; LPS 70 ± 23 mmHg), being reversed by atorvastatin (105 ± 3 mmHg) (p < 0.05). LPS also increased macromolecular leak measured after 100 mg kg(-1) of i.v FITC-BSA (arbitrary grey level adjacent to venules), which again was attenuated by atorvastatin (control 1.9 ± 4.0; LPS 12.0 ± 2.4; LPS + atorvastatin 4.5 ± 2.2) (p < 0.05). Furthermore, immunohistochemistry identified that atorvastatin decreased LPS-induced upregulation of endothelial cell NOSII expression, but NOSIII was unchanged in all groups. Atorvastatin improved MAP and reduced microvascular inflammation during endotoxaemia, associated with a reduction of pro-inflammatory NOSII. This differs from previous studies, whereby pravastatin increased expression of NOSIII. Thus preoperative statins have beneficial anti-inflammatory effects during endotoxaemia, but careful consideration must be given to the specific statin being used.
Asunto(s)
Antiinflamatorios no Esteroideos/uso terapéutico , Atorvastatina/uso terapéutico , Endotoxemia/complicaciones , Microvasos/efectos de los fármacos , Óxido Nítrico Sintasa de Tipo II/metabolismo , Vasculitis/prevención & control , Animales , Atorvastatina/administración & dosificación , Permeabilidad Capilar/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/enzimología , Endotelio Vascular/fisiopatología , Endotoxemia/enzimología , Endotoxemia/fisiopatología , Expresión Génica/efectos de los fármacos , Microscopía Intravital , Leucocitos/enzimología , Leucocitos/fisiología , Lipopolisacáridos/toxicidad , Masculino , Microcirculación/efectos de los fármacos , Microvasos/enzimología , Óxido Nítrico Sintasa de Tipo II/genética , Ratas Wistar , Vasculitis/inducido químicamente , Vasculitis/enzimología , Vasculitis/fisiopatologíaRESUMEN
BACKGROUND: Therapeutic strategies to modulate the host response to bacterial pneumonia are needed to improve outcomes during community-acquired pneumonia. This study used mice with impaired Fas signalling to examine susceptibility to pneumococcal pneumonia and decoy receptor 3 analogue (DcR3-a) to correct factors associated with increased susceptibility. METHODS: Wild-type mice and those with varying degrees of impairment of Fas (lpr) or Fas ligand signalling (gld) were challenged with Streptococcus pneumoniae and microbiological and immunological outcomes measured in the presence or absence of DcR3-a. RESULTS: During established pneumonia, neutrophils became the predominant cell in the airway and gld mice were less able to clear bacteria from the lungs, demonstrating localised impairment of pulmonary neutrophil function in comparison to lpr or wild-type mice. T-cells from gld mice had enhanced activation and reduced apoptosis in comparison to wild-type and lpr mice during established pneumonia. Treatment with DcR3-a reduced T-cell activation and corrected the defect in pulmonary bacterial clearance in gld mice. CONCLUSIONS: The results suggest that imbalance in tumour necrosis factor superfamily signalling and excessive T-cell activation can impair bacterial clearance in the lung but that DcR3-a treatment can reduce T-cell activation, restore optimal pulmonary neutrophil function and enhance bacterial clearance during S pneumoniae infection.
Asunto(s)
Proteína Ligando Fas/metabolismo , Neutrófilos/inmunología , Fagocitos/inmunología , Neumonía Neumocócica/inmunología , Miembro 6b de Receptores del Factor de Necrosis Tumoral/farmacología , Animales , Modelos Animales de Enfermedad , Proteína Ligando Fas/farmacología , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Neutrófilos/efectos de los fármacos , Fagocitos/efectos de los fármacos , Neumonía Neumocócica/metabolismo , Neumonía Neumocócica/microbiología , Neumonía Neumocócica/terapia , Síndrome de Dificultad Respiratoria/inmunología , Síndrome de Dificultad Respiratoria/prevención & control , Transducción de Señal/efectos de los fármacos , Streptococcus pneumoniae/inmunologíaRESUMEN
Sepsis may be modeled using lipopolysaccharide (LPS), which alters levels of nitric oxide (NO), synthesized via endothelial and inducible nitric oxide synthase (eNOS and iNOS). This study aimed to determine whether the Rho kinase (ROCK) inhibitor fasudil protected against LPS-induced (endotoxemia) macromolecular leak and leukocyte adhesion via NOS pathways. Male Wistar rats (283±8g, n=36) were anaesthetized with thiopental and the mesentery prepared for fluorescent intravital microscopy (IVM). Animals received either (i) LPS alone (150µg kg(-1) h(-1) i.v., n=6); (ii) fasudil (FAS, 3mg kg(-1) i.v., n=6) or (iii) fasudil (10mg kg(-1) i.v., n=6), immediately prior to LPS administration, (iv) fasudil (FAS, 3mg kg(-1) i.v., n=6) alone or (v) fasudil (FAS, 10mg kg(-1) i.v., n=6) alone, or (vi) saline alone (1ml kg(-1) h(-1) i.v, n=6) for 4h (240min). LPS increased macromolecular leak (cumulative normalized grey levels, arbitrary units) from post capillary venules (<40µm) and this was reduced by 3mg kg(-1) fasudil, however, 10mg kg(-1) was less effective (t=240min, control: 3.3±1.7; LPS: 15.1±2.0; LPS+3mg kg(-1) fasudil: 3.3±1.1 (p<0.05), LPS+10mg kg(-1) fasudil: 8.4±3.2 NS). The numbers of leukocytes adhering for >1min/100µm venule were reduced by fasudil (t=240min, control: 1.8±0.7; LPS: 7.0±1.0; LPS+3mg kg(-1) fasudil: 1.75±0.25, p<0.05; LPS+10mg kg(-1) fasudil: 1.8±0.8, p<0.05). Immunohistochemistry demonstrated that fasudil increased endothelial cell expression of eNOS during sepsis, and decreased LPS-induced up-regulation of iNOS. Inhibition of ROCK in rats increases eNOS and decreases iNOS during endotoxemia, concomitantly reducing microvascular inflammation. Thus, targeting the ROCK pathway during sepsis could have therapeutic potential for reducing inflammation via a NO dependent mechanism.
Asunto(s)
Endotoxemia/metabolismo , Microcirculación/fisiología , Microvasos/metabolismo , Óxido Nítrico Sintasa/metabolismo , Quinasas Asociadas a rho/metabolismo , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Animales , Presión Sanguínea/efectos de los fármacos , Permeabilidad Capilar/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Endotoxemia/inducido químicamente , Endotoxemia/fisiopatología , Inflamación/inducido químicamente , Inflamación/metabolismo , Inflamación/fisiopatología , Leucocitos/efectos de los fármacos , Leucocitos/metabolismo , Leucocitos/patología , Lipopolisacáridos/farmacología , Masculino , Microcirculación/efectos de los fármacos , Microvasos/efectos de los fármacos , Microvasos/fisiopatología , Óxido Nítrico/sangre , Óxido Nítrico Sintasa de Tipo II/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Ratas , Ratas Wistar , Circulación Esplácnica/efectos de los fármacos , Circulación Esplácnica/fisiología , Quinasas Asociadas a rho/antagonistas & inhibidoresRESUMEN
The response to hemodynamic force is implicated in a number of pathologies including collateral vessel development. However, the transcriptional effect of hemodynamic force is extremely challenging to examine in vivo in mammals without also detecting confounding processes such as hypoxia and ischemia. We therefore serially examined the transcriptional effect of preventing cardiac contraction in zebrafish embryos which can be deprived of circulation without experiencing hypoxia since they obtain sufficient oxygenation by diffusion. Morpholino antisense knock-down of cardiac troponin T2 (tnnt2) prevented cardiac contraction without affecting vascular development. Gene expression in whole embryo RNA from tnnt2 or control morphants at 36, 48, and 60 h postfertilization (hpf) was assessed using Affymetrix GeneChip Zebrafish Genome Arrays (>14,900 transcripts). We identified 308 differentially expressed genes between tnnt2 and control morphants. One such (CXCR4a) was significantly more highly expressed in tnnt2 morphants at 48 and 60 hpf than controls. In situ hybridization localized CXCR4a upregulation to endothelium of both tnnt2 morphants and gridlock mutants (which have an occluded aorta preventing distal blood flow). This upregulation appears to be of functional significance as either CXCR4a knock-down or pharmacologic inhibition impaired the ability of gridlock mutants to recover blood flow via collateral vessels. We conclude absence of hemodynamic force induces endothelial CXCR4a upregulation that promotes recovery of blood flow.
Asunto(s)
Embrión no Mamífero/metabolismo , Perfilación de la Expresión Génica , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Receptores CXCR4/genética , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Animales , Arteriolas/crecimiento & desarrollo , Velocidad del Flujo Sanguíneo , Análisis por Conglomerados , Circulación Colateral , Regulación hacia Abajo , Embrión no Mamífero/embriología , Femenino , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen/métodos , Hibridación in Situ , Masculino , Contracción Miocárdica , Oligonucleótidos Antisentido , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Troponina T/genética , Pez Cebra/embriologíaRESUMEN
BACKGROUND: Developing vectors that target specifically to disease sites after systemic injection is an important goal in gene therapy research. METHODS: We prepared fluorescent DNA polyplexes (< or =150 nm in diameter) comprising plasmid DNA condensed with poly(L-lysine) and coated with a multivalent reactive copolymer based on poly[N-(2-hydroxypropyl)methacrylamide] (pHPMA). These polyplexes were then surface modified with a recombinant P-selectin glycoprotein ligand-1 immunoglobulin chimera (rPSGL-Ig) previously investigated as a selectin antagonist in clinical studies. RESULTS: Five minutes after jugular vein injection of these polyplexes, fluorescence accumulation in inflamed cremasteric venules of C57BL6 mice was more than eight-fold higher than that observed after injection of Fc-blocked control polyplexes. Fluorescence above background was not observed in P-selectin deficient mice, confirming the specificity for P-selectin in this model. CONCLUSIONS: These data provide encouragement for the further development of rPSGL-Ig-coated polyplexes as potential nonviral vectors for targeted gene therapy in inflammatory conditions, such as ischaemia reperfusion injury, unstable atherosclerotic plaques and myocarditis. This approach may also be transferable to the use of other targeting ligands whose cognate partner is specifically upregulated on the vascular endothelium in individual pathological situations.
Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , Endotelio/patología , Inflamación/tratamiento farmacológico , Glicoproteínas de Membrana/administración & dosificación , Selectina-P/metabolismo , Polímeros/química , Animales , Colorantes Fluorescentes , Inmunoglobulinas , Glicoproteínas de Membrana/farmacocinética , Ratones , Ratones Endogámicos C57BL , Microscopía , Plásmidos , Polilisina , Polímeros/farmacocinética , Proteínas RecombinantesRESUMEN
Ly-6G is a member of the Ly-6 family of GPI-linked proteins, which is expressed on murine neutrophils. Antibodies against Ly-6G cause neutropenia, and fatal reactions also develop if mice are primed with TNF-alpha prior to antibody treatment. We have investigated the mechanisms behind these responses to Ly-6G ligation in the belief that similar mechanisms may be involved in neutropenia and respiratory disorders associated with alloantibody ligation of the related Ly-6 family member, NB1, in humans. Neutrophil adhesion, microvascular obstruction, breathing difficulties, and death initiated by anti-Ly-6G antibodies in TNF-alpha-primed mice were shown to be highly complement-dependent, partly mediated by CD11b, CD18, and FcgammaR and associated with clustering of Ly-6G. Neutrophil depletion, on the other hand, was only partly complement-dependent and was not altered by blockade of CD11b, CD18, or FcgammaR. Unlike other neutrophil-activating agents, Ly-6G ligation did not induce neutropenia via sequestration in the lungs. Cross-linking Ly-6G mimicked the responses seen with whole antibody in vivo and also activated murine neutrophils in vitro. Although this suggests that the responses are, in part, mediated by nonspecific properties of antibody ligation, neutrophil depletion requires an additional mechanism possibly specific to the natural function of Ly-6G.
Asunto(s)
Antígenos Ly/inmunología , Proteínas del Sistema Complemento/inmunología , Neutropenia/inmunología , Neutrófilos/inmunología , Insuficiencia Respiratoria/inmunología , Animales , Anticuerpos Monoclonales/farmacología , Antígeno CD11b/inmunología , Antígenos CD18/inmunología , Reactivos de Enlaces Cruzados , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microcirculación/inmunología , Neutropenia/mortalidad , Neutropenia/fisiopatología , Receptores de IgG/genética , Receptores de IgG/inmunología , Respiración , Insuficiencia Respiratoria/mortalidad , Insuficiencia Respiratoria/fisiopatología , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
RATIONALE: The role of NADPH oxidase activation in pneumonia is complex because reactive oxygen species contribute to both microbial killing and regulation of the acute pulmonary infiltrate. The relative importance of each role remains poorly defined in community-acquired pneumonia. OBJECTIVES: We evaluated the contribution of NADPH oxidase-derived reactive oxygen species to the pathogenesis of pneumococcal pneumonia, addressing both the contribution to microbial killing and regulation of the inflammatory response. METHODS: Mice deficient in the gp91(phox) component of the phagocyte NADPH oxidase were studied after pneumococcal challenge. MEASUREMENTS AND MAIN RESULTS: gp91(phox)(-/-) mice demonstrated no defect in microbial clearance as compared with wild-type C57BL/6 mice. A significant increase in bacterial clearance from the lungs of gp91(phox)(-/-) mice was associated with increased numbers of neutrophils in the lung, lower rates of neutrophil apoptosis, and enhanced activation. Marked alterations in pulmonary cytokine/chemokine expression were also noted in the lungs of gp91(phox)(-/-) mice, characterized by elevated levels of tumor necrosis factor-alpha, KC, macrophage inflammatory protein-2, monocyte chemotactic protein-1, and IL-6. The greater numbers of neutrophils in gp91(phox)(-/-) mice were not associated with increased lung injury. Levels of neutrophil elastase in bronchoalveolar lavage were not decreased in gp91(phox)(-/-) mice. CONCLUSIONS: During pneumococcal pneumonia, NADPH oxidase-derived reactive oxygen species are redundant for host defense but limit neutrophil recruitment and survival. Decreased NADPH oxidase-dependent reactive oxygen species production is well tolerated and improves disease outcome during pneumococcal pneumonia by removing neutrophils from the tight constraints of reactive oxygen species-mediated regulation.
Asunto(s)
Glicoproteínas de Membrana/deficiencia , NADPH Oxidasas/inmunología , Neutrófilos/inmunología , Neumonía Neumocócica/inmunología , Animales , Modelos Animales de Enfermedad , Femenino , Inflamación , Glicoproteínas de Membrana/inmunología , Ratones , Ratones Noqueados , NADPH Oxidasa 2 , NADPH Oxidasas/deficiencia , Infiltración Neutrófila , Neutrófilos/microbiología , Neumonía Neumocócica/fisiopatología , Especies Reactivas de Oxígeno/inmunologíaRESUMEN
RATIONALE: Cystic fibrosis lung disease is characterized by accumulation of apoptotic neutrophils, indicating impaired clearance of dying cells. Pseudomonas aeruginosa, the principal microbial pathogen in cystic fibrosis, manipulates apoptosis induction via production of toxic metabolites. Whether these metabolites, particularly pyocyanin, can also modulate apoptotic cell engulfment is unknown. OBJECTIVES: To assess the effects of pyocyanin on apoptotic cell engulfment by macrophages in vitro and in vivo and to investigate potential mechanisms of the observed effects. METHODS: Human monocyte-derived macrophages were treated with pyocyanin before challenge with apoptotic neutrophils, apoptotic Jurkat cells, or latex beads, and phagocytosis was assessed by light microscopy and flow cytometry. Effects of pyocyanin production on apoptotic cell clearance in vivo were assessed in a murine model, comparing infection by wild-type or pyocyanin-deficient P. aeruginosa. Oxidant production was investigated using fluorescent probes and pharmacologic inhibition and Rho GTPase signaling by immunoblotting and inhibitor studies. MEASUREMENTS AND MAIN RESULTS: Pyocyanin treatment impaired macrophage engulfment of apoptotic cells in vitro, without inducing significant macrophage apoptosis, whereas latex bead uptake was preserved. Macrophage ingestion of apoptotic cells was reduced and late apoptotic/necrotic cells were increased in mice infected with pyocyanin-producing P. aeruginosa compared with the pyocyanin-deficient strain. Inhibition of apoptotic cell uptake involved intracellular generation of reactive oxygen species (ROS) and effects on Rho GTPase signaling. Antioxidants or blockade of Rho signaling substantially restored apoptotic cell engulfment. CONCLUSIONS: These studies demonstrate that P. aeruginosa can manipulate the inflammatory microenvironment through inhibition of apoptotic cell engulfment, and suggest potential strategies to limit pulmonary inflammation in cystic fibrosis.
Asunto(s)
Apoptosis/efectos de los fármacos , Fibrosis Quística/inmunología , Macrófagos Alveolares/efectos de los fármacos , Fagocitosis/efectos de los fármacos , Neumonía Bacteriana/inmunología , Pseudomonas aeruginosa/metabolismo , Piocianina/farmacología , Animales , Humanos , Etiquetado Corte-Fin in Situ , Células Jurkat , Macrófagos Alveolares/inmunología , Ratones , Ratones Endogámicos C57BL , Microesferas , Proteínas de Unión al GTP Monoméricas/metabolismo , Fagocitosis/inmunología , Pseudomonas aeruginosa/inmunología , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
OBJECTIVE: The role of ischemia in collateral vessel development (arteriogenesis) is a contentious issue that cannot be addressed using mammalian models. To investigate this, we developed models of arteriogenesis using the zebrafish embryo, which gains sufficient oxygenation via diffusion to prevent ischemia in response to arterial occlusion. METHODS AND RESULTS: We studied gridlock mutant embryos that suffer a permanently occluded aorta and show that these restore aortic blood flow by collateral vessels. We phenocopied gridlock mutants by laser-induced proximal aortic occlusion in transgenic Fli1:eGFP/GATA1:dsRED embryos. Serial imaging showed these restore aortic blood flow via collateral vessels by recruitment of preexisting endothelium in a manner similar to gridlocks. Collateral aortic blood flow in gridlock mutants was dependent on both nitric oxide and myeloid cells. Confocal microscopy of transgenic gridlock/Fli1:eGFP mutants demonstrated no aberrant angiogenic response to the aortic occlusion. qPCR of HIF1alpha expression confirmed the absence of hypoxia in this model system. CONCLUSIONS: We conclude that NO and myeloid cell-dependent collateral vessel development is an evolutionarily ancient response to arterial occlusion and is able to proceed in the absence of ischemia.
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Enfermedades de la Aorta/fisiopatología , Arteriopatías Oclusivas/fisiopatología , Arterias/crecimiento & desarrollo , Circulación Colateral , Isquemia/fisiopatología , Neovascularización Fisiológica , Pez Cebra/embriología , Animales , Animales Modificados Genéticamente , Enfermedades de la Aorta/embriología , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/metabolismo , Arteriopatías Oclusivas/embriología , Arteriopatías Oclusivas/genética , Arteriopatías Oclusivas/metabolismo , Arterias/embriología , Arterias/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Hipoxia de la Célula , Modelos Animales de Enfermedad , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Isquemia/embriología , Isquemia/genética , Isquemia/metabolismo , Microscopía Confocal , Mutación , Células Mieloides/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa/metabolismo , Proteína Proto-Oncogénica c-fli-1/genética , Proteína Proto-Oncogénica c-fli-1/metabolismo , ARN Mensajero/metabolismo , Factores de Tiempo , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
The pulmonary innate response to low-dose bacterial challenge requires functioning alveolar macrophages (AM) but also subsequent macrophage apoptosis. To address the role of reactive oxygen species (ROS) and nitric oxide (NO) in AM apoptosis, sub-clinical Streptococcus pneumoniae infection was established in gp91(phox-/-) and inducible NO synthase deficient (iNOS(-/-)) mice. Both AM apoptosis and the number of macrophages containing apoptotic bodies are reduced in iNOS(-/-) as compared to control or gp91(phox-/-) mice. iNOS(-/-) mice recruit neutrophils and generate TNF-alpha to compensate for impaired AM competence but ROS deficiency has no apparent effect on AM function in this model.
Asunto(s)
Pulmón/microbiología , Macrófagos Alveolares/inmunología , Óxido Nítrico/inmunología , Infecciones Neumocócicas/inmunología , Especies Reactivas de Oxígeno/inmunología , Streptococcus pneumoniae/inmunología , Animales , Apoptosis/inmunología , Femenino , Inmunidad Innata/inmunología , Macrófagos Alveolares/metabolismo , Glicoproteínas de Membrana/deficiencia , Ratones , Ratones Endogámicos C57BL , NADPH Oxidasa 2 , NADPH Oxidasas/deficiencia , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo II/deficiencia , Infecciones Neumocócicas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Streptococcus pneumoniae/metabolismoRESUMEN
Regulation of the inflammatory infiltrate is critical to the successful outcome of pneumonia. Alveolar macrophage apoptosis is a feature of pneumococcal infection and aids disease resolution. The host benefits of macrophage apoptosis during the innate response to bacterial infection are incompletely defined. Because NO is required for optimal macrophage apoptosis during pneumococcal infection, we have explored the role of macrophage apoptosis in regulating inflammatory responses during pneumococcal pneumonia, using inducible NO synthase (iNOS)-deficient mice. iNOS(-/-) mice demonstrated decreased numbers of apoptotic macrophages as compared with wild-type C57BL/6 mice following pneumococcal challenge, greater recruitment of neutrophils to the lung and enhanced expression of TNF-alpha. Pharmacologic inhibition of iNOS produced similar results. Greater pulmonary inflammation was associated with greater levels of early bacteremia, IL-6 production, lung inflammation, and mortality within the first 48 h in iNOS(-/-) mice. Labeled apoptotic alveolar macrophages were phagocytosed by resident macrophages in the lung and intratracheal instillation of exogenous apoptotic macrophages decreased neutrophil recruitment in iNOS(-/-) mice and decreased TNF-alpha mRNA in lungs and protein in bronchial alveolar lavage, as well as chemokines and cytokines including IL-6. These changes were associated with a lower probability of mice becoming bacteremic. This demonstrates the potential of apoptotic macrophages to down-regulate the inflammatory response and for the first time in vivo demonstrates that clearance of apoptotic macrophages decreases neutrophil recruitment and invasive bacterial disease during pneumonia.
Asunto(s)
Apoptosis , Pulmón/inmunología , Macrófagos Alveolares/fisiología , Óxido Nítrico Sintasa de Tipo II/deficiencia , Neumonía Neumocócica/inmunología , Animales , Bacteriemia/enzimología , Bacteriemia/genética , Bacteriemia/inmunología , Biomarcadores/análisis , Modelos Animales de Enfermedad , Pulmón/enzimología , Pulmón/microbiología , Macrófagos Alveolares/enzimología , Ratones , Ratones Mutantes , Neutrófilos/enzimología , Neutrófilos/inmunología , Óxido Nítrico Sintasa de Tipo II/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo II/genética , Neumonía Neumocócica/enzimología , Neumonía Neumocócica/genética , Streptococcus pneumoniae/inmunología , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Clearance of neutrophils from inflamed sites is critical for resolution of inflammation, but pathogen-driven neutrophil apoptosis can impair host defenses. We previously showed that pyocyanin, a phenazine toxic metabolite produced by Pseudomonas aeruginosa, accelerates neutrophil apoptosis in vitro. We compared wild-type and pyocyanin-deficient strains of P. aeruginosa in a murine model of acute pneumonia. Intratracheal instillation of either strain of P. aeruginosa caused a rapid increase in bronchoalveolar lavage neutrophil counts up to 18 h after infection. In wild-type infection, neutrophil numbers then declined steadily, whereas neutrophil numbers increased up to 48 h in mice infected with pyocyanin-deficient P. aeruginosa. In keeping with these differences, pyocyanin production was associated with reduced bacterial clearance from the lungs. Neutrophil apoptosis was increased in mice infected with wild-type compared with the phenazine-deficient strain or two further strains that lack pyocyanin production, but produce other phenazines. Concentrations of potent neutrophil chemokines (MIP-2, KC) and cytokines (IL-6, IL-1beta) were significantly lower in wild-type compared with phenazine-deficient strain-infected mice at 18 h. We conclude that pyocyanin production by P. aeruginosa suppresses the acute inflammatory response by pathogen-driven acceleration of neutrophil apoptosis and by reducing local inflammation, and that this is advantageous for bacterial survival.
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Neutrófilos/inmunología , Neutrófilos/patología , Neumonía Bacteriana/inmunología , Neumonía Bacteriana/patología , Infecciones por Pseudomonas/inmunología , Infecciones por Pseudomonas/patología , Pseudomonas aeruginosa/patogenicidad , Piocianina/biosíntesis , Animales , Apoptosis , Líquido del Lavado Bronquioalveolar/citología , Líquido del Lavado Bronquioalveolar/inmunología , Quimiocinas/biosíntesis , Citocinas/biosíntesis , Ratones , Ratones Endogámicos C57BL , Mutación , Fenazinas/metabolismo , Neumonía Bacteriana/microbiología , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/inmunología , Pseudomonas aeruginosa/metabolismo , Piocianina/genéticaRESUMEN
P-selectin glycoprotein ligand-1 (PSGL-1) binding to P-selectin controls early leukocyte rolling during inflammation. Interestingly, antibodies and pharmacological inhibitors (eg, rPSGL-Ig) that target the N-terminus of PSGL-1 reduce but do not abolish P-selectin-dependent leukocyte rolling in vivo whereas PSGL-1-deficient mice have almost no P-selectin-dependent rolling. We have investigated mechanisms of P-selectin-dependent, PSGL-1-independent rolling using intravital microscopy. Initially we used fluorescent microspheres to study the potential of L-selectin and the minimal selectin ligand sialyl Lewis(x) (sLe(x)) to interact with postcapillary venules in the absence of PSGL-1. Microspheres coated with combinations of L-selectin and sLe(x) interacted with surgically stimulated cremaster venules in a P-selectin-dependent manner. Microspheres coated with either L-selectin or sLe(x) alone showed less evidence of interaction. We also investigated leukocyte rolling in the presence of PSGL-1 antibody or inhibitor (rPSGL-Ig), both of which partially inhibited P-selectin-dependent leukocyte rolling. Residual rolling was substantially inhibited by L-selectin-blocking antibody or a previously described sLe(x) mimetic (CGP69669A). Together these data suggest that leukocytes can continue to roll in the absence of optimal P-selectin/PSGL-1 interaction using an alternative mechanism that involves P-selectin-, L-selectin-, and sLe(x)-bearing ligands.
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Selectina L/metabolismo , Rodamiento de Leucocito , Leucocitos/citología , Glicoproteínas de Membrana/metabolismo , Selectina-P/metabolismo , Animales , Biotinilación , Adhesión Celular , Técnicas de Cultivo , Glicoproteínas/metabolismo , Leucocitos/metabolismo , Ligandos , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía , Microesferas , Modelos Biológicos , Oligosacáridos/metabolismo , Unión Proteica , Antígeno Sialil Lewis X , Factores de TiempoRESUMEN
Macrophages are critical effectors of bacterial clearance and must retain viability, despite exposure to toxic bacterial products, until key antimicrobial functions are performed. Subsequently, host-mediated macrophage apoptosis aids resolution of infection. The ability of macrophages to make this transition from resistance to susceptibility to apoptosis is important for effective host innate immune responses. We investigated the role of Mcl-1, an essential regulator of macrophage lifespan, in this switch from viability to apoptosis, using the model of pneumococcal-associated macrophage apoptosis. Upon exposure to pneumococci, macrophages initially upregulate Mcl-1 protein and maintain viability for up to 14 hours. Subsequently, macrophages reduce expression of full-length Mcl-1 and upregulate a 34-kDa isoform of Mcl-1 corresponding to a novel BH3-only splice variant, Mcl-1(Exon-1). Change in expression of Mcl-1 protein is associated with mitochondrial membrane permeabilization, which is characterized by loss of mitochondrial inner transmembrane potential and translocation of cytochrome c and apoptosis-inducing factor. Following pneumococcal infection, macrophages expressing full-length human Mcl-1 as a transgene exhibit a delay in apoptosis and in bacterial killing. Mcl-1 transgenic mice clear pneumococci from the lung less efficiently than nontransgenic mice. Dynamic changes in Mcl-1 expression determine macrophage viability as well as antibacterial host defense.
Asunto(s)
Apoptosis , Macrófagos/metabolismo , Proteínas de Neoplasias/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Streptococcus pneumoniae , Empalme Alternativo/genética , Empalme Alternativo/fisiología , Animales , Apoptosis/genética , Supervivencia Celular/fisiología , Regulación de la Expresión Génica , Humanos , Macrófagos/microbiología , Macrófagos/patología , Potenciales de la Membrana , Ratones , Ratones Transgénicos , Mitocondrias/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Proteínas de Neoplasias/genética , Neumonía Neumocócica/genética , Neumonía Neumocócica/metabolismo , Neumonía Neumocócica/patología , Isoformas de Proteínas/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/genéticaRESUMEN
Selectins and their ligands support leukocyte rolling, facilitating the subsequent firm adhesion and migration that occur during inflammation. TBC-1269 (Bimosiamose), a structural mimetic of natural selectin ligands, inhibits P-, E-, and L-selectin in vitro, has anti-inflammatory effects in vivo, and recently underwent phase II clinical trials for childhood asthma and psoriasis. We studied whether the anti-inflammatory effects of TBC-1269 could be related to leukocyte rolling in vivo. Although TBC-1269 inhibited rolling of a murine leukocyte cell line on murine P-selectin in vitro and thioglycollate-induced peritonitis in vivo, it did not alter leukocyte rolling in mouse cremaster venules. TBC-1269 reduced neutrophil recruitment in thioglycollate-induced peritonitis in wild-type and P-selectin-/- mice but not in E-selectin-/- mice. We suggest that the in vivo effects of TBC-1269 may be mediated through E-selectin but do not appear to involve leukocyte rolling.
Asunto(s)
Compuestos de Bifenilo/uso terapéutico , Selectina E/fisiología , Inflamación/patología , Leucocitos/metabolismo , Manósidos/uso terapéutico , Imitación Molecular , Selectina-P/fisiología , Animales , Unión Competitiva , Selectina E/genética , Selectina E/inmunología , Leucocitos/inmunología , Leucocitos/patología , Ligandos , Masculino , Manosa/análogos & derivados , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Neutrófilos/inmunología , Neutrófilos/metabolismo , Neutrófilos/patología , Selectina-P/genética , Selectina-P/inmunología , Peptoides/química , Peptoides/farmacología , Peritonitis/inducido químicamente , Peritonitis/patología , Tioglicolatos/toxicidad , Vénulas/citologíaRESUMEN
The regulation of systemic and local neutrophil activation is crucial to the clearance of infections and the successful resolution of inflammation without progress to tissue damage or disseminated inflammatory reactions. Using purified lipopolysaccharide (pLPS) and highly purified neutrophils, we have previously shown that Toll-like receptor 4 signaling is a potent neutrophil activator, but a poor stimulator of survival. In the presence of peripheral blood mononuclear cells (PBMCs), however, pLPS becomes a potent neutrophil survival factor. Interleukin (IL)-1beta has been identified as an important neutrophil activator and prosurvival cytokine, and is produced in abundance by LPS-stimulated PBMCs. We now show that IL-1beta fails to activate highly purified neutrophils or enhance their survival, but in the presence of PBMCs, IL-1beta induces neutrophil survival. We hypothesized that LPS-primed neutrophils might become responsive to IL-1beta, but were unable to demonstrate this. Moreover, IL-1ra failed to prevent pLPS + PBMC-dependent neutrophil survival. In studies of IL-1R1(-/-) mice, we found that LPS was still able to mediate neutrophil survival, and neutrophil survival was enhanced by the addition of monocytic cells. Thus an important paradigm of neutrophil regulation needs to be viewed in the context of a cellular network in which actions of IL-1beta on neutrophils are indirect and mediated by other cells.
Asunto(s)
Interleucina-1/fisiología , Glicoproteínas de Membrana/metabolismo , Neutrófilos/metabolismo , Receptores de Superficie Celular/metabolismo , Animales , Apoptosis , Antígeno CD11b/biosíntesis , Adhesión Celular , Supervivencia Celular , Citocinas/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Inflamación , Interleucina-1/biosíntesis , Interleucina-1/metabolismo , Selectina L/biosíntesis , Leucocitos Mononucleares/metabolismo , Lipopolisacáridos/química , Ratones , Ratones Transgénicos , Modelos Biológicos , Activación Neutrófila , Transducción de Señal , Factores de Tiempo , Receptor Toll-Like 4 , Receptores Toll-LikeRESUMEN
Leptin, the satiety hormone, appears to act as a link between nutritional status and immune function. It has been shown to elicit a number of immunoregulatory effects, including the promotion of T cell proliferative responses, and the induction of proinflammatory cytokines. Leptin deficiency is associated with an increased susceptibility to infection. As polymorphonuclear neutrophils (PMN) play a major role in innate immunity and host defense against infection, this study evaluated the influence of leptin on PMN activation. The presence of leptin receptor in human PMN was determined both at mRNA and protein levels, and the effect of leptin on PMN activation, as assessed by CD11b expression, was evaluated using flow cytometry. In contrast to monocytes, which express both the short and long forms of the leptin receptor (Ob-Ra and Ob-Rb, respectively), PMN expressed only Ob-Ra. Leptin up-regulated the expression of CD11b, an early marker of PMN activation, on PMN in whole blood, yet it had no effect on purified PMN, even those treated by submaximal doses of TNF-alpha or PMA. The kinetics of leptin-induced activation in whole blood were consistent with an indirect effect mediated by monocytes, and 71% of the leptin-stimulatory effect on PMN was blocked by a TNF-alpha inhibitor. Leptin-mediated induction of CD11b expression was observed when purified PMN were coincubated with purified monocytes. In conclusion, although leptin activates PMN, it does so indirectly via TNF-alpha release from monocytes. These findings provide an additional link among the obesity-derived hormone leptin, innate immune function, and infectious disease.
Asunto(s)
Leptina/fisiología , Activación Neutrófila/inmunología , Factor de Necrosis Tumoral alfa/biosíntesis , Antígeno CD11b/biosíntesis , Antígeno CD11b/sangre , Comunicación Celular/inmunología , Separación Celular , Citometría de Flujo , Regulación de la Expresión Génica/inmunología , Humanos , Leptina/sangre , Monocitos/inmunología , Monocitos/metabolismo , Neutrófilos/inmunología , Neutrófilos/metabolismo , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/genética , Receptores de Superficie Celular/biosíntesis , Receptores de Superficie Celular/genética , Receptores de Leptina , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/fisiologíaRESUMEN
Selectins mediate leukocyte rolling and may represent good anti-inflammatory drug targets. Detailed knowledge regarding the structure of selectin ligands has permitted development of selectin antagonists with varying specificities and activity. Efficacy of monovalent selectin antagonists may be increased by presenting them on a polymer backbone. We have synthesized a range of multivalent selectin antagonists and characterized their activity by using intravital microscopy of the mouse cremaster muscle. The monovalent inhibitor CGP77175A inhibited E-selectin-dependent leukocyte rolling at a dose of 3 mg/kg. Multivalent presentation of CGP77175A on a modified polylysine backbone (degree of polymerization = 1200; 50% of the polylysines carry the inhibitor) greatly enhanced in vivo activity giving an inhibitor that produced an equivalent effect at 0.1 mg/kg. The polylysine conjugate was also longer acting than the monovalent antagonist. In spite of greatly enhanced activity against E-selectin compared with monovalent inhibitor, the multivalent inhibitor had no measurable effect on P- or L-selectin-dependent leukocyte rolling.
Asunto(s)
Selectina E/metabolismo , Rodamiento de Leucocito/efectos de los fármacos , Oligosacáridos/química , Polímeros/farmacología , Animales , Cinética , Ratones , Oligosacáridos/farmacología , Polilisina/química , Polilisina/farmacología , Polímeros/química , Antígeno Sialil Lewis XRESUMEN
The role of alveolar macrophages (AM) in host defense against pulmonary infection has been difficult to establish using in vivo models. This may reflect a reliance on models of fulminant infection. To establish a unique model of resolving infection, with which to address the function of AM, C57BL/6 mice received low-dose intratracheal administration of pneumococci. Administration of low doses of pneumococci produced a resolving model of pulmonary infection characterized by clearance of bacteria without features of pneumonia. AM depletion in this model significantly increased bacterial outgrowth in the lung. Interestingly, a significant increase in the number of apoptotic AM was noted with the low-dose infection as compared with mock infection. Caspase inhibition in this model decreased AM apoptosis and increased the number of bacteremic mice, indicating a novel role for caspase activation in pulmonary innate defense against pneumococci. These results suggest that AM play a key role in clearance of bacteria from the lung during subclinical infection and that induction of AM apoptosis contributes to the microbiologic host defense against pneumococci.
Asunto(s)
Apoptosis/inmunología , Modelos Animales de Enfermedad , Macrófagos Alveolares/inmunología , Macrófagos Alveolares/microbiología , Neumonía Neumocócica/inmunología , Neumonía Neumocócica/microbiología , Clorometilcetonas de Aminoácidos/administración & dosificación , Animales , Bacteriemia/enzimología , Bacteriemia/inmunología , Bacteriemia/microbiología , Inhibidores de Caspasas , Recuento de Células , Inhibidores de Cisteína Proteinasa/administración & dosificación , Relación Dosis-Respuesta Inmunológica , Femenino , Inmunidad Innata , Inyecciones Intraperitoneales , Intubación Intratraqueal , Macrófagos Alveolares/enzimología , Macrófagos Alveolares/patología , Ratones , Ratones Endogámicos C57BL , Neumonía Neumocócica/enzimología , Neumonía Neumocócica/patología , Streptococcus pneumoniae/crecimiento & desarrollo , Streptococcus pneumoniae/inmunología , Regulación hacia Arriba/inmunologíaRESUMEN
Widespread microvascular injury followed by vessel obstruction may lead to disseminated intravascular coagulation (DIC). We describe a murine model wherein leukocytes interacting with inflamed microvessels in vivo are activated by antibodies. Treatment of tumor necrosis factor alpha (TNF-alpha)-primed mice with anti-Ly-6G antibodies reproduced many of the features of septic or traumatic shock including microvessel obstruction and coagulation, severe vasculitis, respiratory difficulties, and vascular leakage. Mice lacking either E-selectin or P-selectin were protected from this reaction as were animals treated with a combination of either selectin-blocking antibodies and heparin or a selectin antagonist plus heparin. Combined blockade of leukocyte/platelet adhesion and coagulation may provide convincing protection in DIC.