RESUMO
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.
Assuntos
Proteína Ligante Fas/metabolismo , Neutrófilos/imunologia , Fagócitos/imunologia , Pneumonia Pneumocócica/imunologia , Membro 6b de Receptores do Fator de Necrose Tumoral/farmacologia , Animais , Modelos Animais de Doenças , Proteína Ligante Fas/farmacologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Neutrófilos/efeitos dos fármacos , Fagócitos/efeitos dos fármacos , Pneumonia Pneumocócica/metabolismo , Pneumonia Pneumocócica/microbiologia , Pneumonia Pneumocócica/terapia , Síndrome do Desconforto Respiratório/imunologia , Síndrome do Desconforto Respiratório/prevenção & controle , Transdução de Sinais/efeitos dos fármacos , Streptococcus pneumoniae/imunologiaRESUMO
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.
Assuntos
Endotoxemia/metabolismo , Microcirculação/fisiologia , Microvasos/metabolismo , Óxido Nítrico Sintase/metabolismo , Quinases Associadas a rho/metabolismo , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Animais , Pressão Sanguínea/efeitos dos fármacos , Permeabilidade Capilar/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Endotoxemia/induzido quimicamente , Endotoxemia/fisiopatologia , Inflamação/induzido quimicamente , Inflamação/metabolismo , Inflamação/fisiopatologia , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Leucócitos/patologia , Lipopolissacarídeos/farmacologia , Masculino , Microcirculação/efeitos dos fármacos , Microvasos/efeitos dos fármacos , Microvasos/fisiopatologia , Óxido Nítrico/sangue , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Ratos , Ratos Wistar , Circulação Esplâncnica/efeitos dos fármacos , Circulação Esplâncnica/fisiologia , Quinases Associadas a rho/antagonistas & inibidoresRESUMO
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.
Assuntos
Antígenos Ly/imunologia , Proteínas do Sistema Complemento/imunologia , Neutropenia/imunologia , Neutrófilos/imunologia , Insuficiência Respiratória/imunologia , Animais , Anticorpos Monoclonais/farmacologia , Antígeno CD11b/imunologia , Antígenos CD18/imunologia , Reagentes de Ligações Cruzadas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microcirculação/imunologia , Neutropenia/mortalidade , Neutropenia/fisiopatologia , Receptores de IgG/genética , Receptores de IgG/imunologia , Respiração , Insuficiência Respiratória/mortalidade , Insuficiência Respiratória/fisiopatologia , Fator de Necrose Tumoral alfa/imunologiaRESUMO
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.
Assuntos
Embrião não Mamífero/metabolismo , Perfilação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Receptores CXCR4/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Arteríolas/crescimento & desenvolvimento , Velocidade do Fluxo Sanguíneo , Análise por Conglomerados , Circulação Colateral , Regulação para Baixo , Embrião não Mamífero/embriologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes/métodos , Hibridização In Situ , Masculino , Contração Miocárdica , Oligonucleotídeos Antissenso , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Troponina T/genética , Peixe-Zebra/embriologiaRESUMO
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.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Endotélio/patologia , Inflamação/tratamento farmacológico , Glicoproteínas de Membrana/administração & dosagem , Selectina-P/metabolismo , Polímeros/química , Animais , Corantes Fluorescentes , Imunoglobulinas , Glicoproteínas de Membrana/farmacocinética , Camundongos , Camundongos Endogâmicos C57BL , Microscopia , Plasmídeos , Polilisina , Polímeros/farmacocinética , Proteínas RecombinantesRESUMO
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.
Assuntos
Apoptose/efeitos dos fármacos , Fibrose Cística/imunologia , Macrófagos Alveolares/efeitos dos fármacos , Fagocitose/efeitos dos fármacos , Pneumonia Bacteriana/imunologia , Pseudomonas aeruginosa/metabolismo , Piocianina/farmacologia , Animais , Humanos , Marcação In Situ das Extremidades Cortadas , Células Jurkat , Macrófagos Alveolares/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Microesferas , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Fagocitose/imunologia , Pseudomonas aeruginosa/imunologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
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.
Assuntos
Glicoproteínas de Membrana/deficiência , NADPH Oxidases/imunologia , Neutrófilos/imunologia , Pneumonia Pneumocócica/imunologia , Animais , Modelos Animais de Doenças , Feminino , Inflamação , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Knockout , NADPH Oxidase 2 , NADPH Oxidases/deficiência , Infiltração de Neutrófilos , Neutrófilos/microbiologia , Pneumonia Pneumocócica/fisiopatologia , Espécies Reativas de Oxigênio/imunologiaRESUMO
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.
Assuntos
Apoptose , Macrófagos/metabolismo , Proteínas de Neoplasias/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Streptococcus pneumoniae , Processamento Alternativo/genética , Processamento Alternativo/fisiologia , Animais , Apoptose/genética , Sobrevivência Celular/fisiologia , Regulação da Expressão Gênica , Humanos , Macrófagos/microbiologia , Macrófagos/patologia , Potenciais da Membrana , Camundongos , Camundongos Transgênicos , Mitocôndrias/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides , Proteínas de Neoplasias/genética , Pneumonia Pneumocócica/genética , Pneumonia Pneumocócica/metabolismo , Pneumonia Pneumocócica/patologia , Isoformas de Proteínas/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/genéticaRESUMO
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.
Assuntos
Doenças da Aorta/fisiopatologia , Arteriopatias Oclusivas/fisiopatologia , Artérias/crescimento & desenvolvimento , Circulação Colateral , Isquemia/fisiopatologia , Neovascularização Fisiológica , Peixe-Zebra/embriologia , Animais , Animais Geneticamente Modificados , Doenças da Aorta/embriologia , Doenças da Aorta/genética , Doenças da Aorta/metabolismo , Arteriopatias Oclusivas/embriologia , Arteriopatias Oclusivas/genética , Arteriopatias Oclusivas/metabolismo , Artérias/embriologia , Artérias/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Hipóxia Celular , Modelos Animais de Doenças , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Isquemia/embriologia , Isquemia/genética , Isquemia/metabolismo , Microscopia Confocal , Mutação , Células Mieloides/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/metabolismo , Proteína Proto-Oncogênica c-fli-1/genética , Proteína Proto-Oncogênica c-fli-1/metabolismo , RNA Mensageiro/metabolismo , Fatores de Tempo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
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.
Assuntos
Compostos de Bifenilo/uso terapêutico , Selectina E/fisiologia , Inflamação/patologia , Leucócitos/metabolismo , Manosídeos/uso terapêutico , Mimetismo Molecular , Selectina-P/fisiologia , Animais , Ligação Competitiva , Selectina E/genética , Selectina E/imunologia , Leucócitos/imunologia , Leucócitos/patologia , Ligantes , Masculino , Manose/análogos & derivados , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/imunologia , Neutrófilos/metabolismo , Neutrófilos/patologia , Selectina-P/genética , Selectina-P/imunologia , Peptoides/química , Peptoides/farmacologia , Peritonite/induzido quimicamente , Peritonite/patologia , Tioglicolatos/toxicidade , Vênulas/citologiaRESUMO
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.
Assuntos
Selectina E/metabolismo , Migração e Rolagem de Leucócitos/efeitos dos fármacos , Oligossacarídeos/química , Polímeros/farmacologia , Animais , Cinética , Camundongos , Oligossacarídeos/farmacologia , Polilisina/química , Polilisina/farmacologia , Polímeros/química , Antígeno Sialil Lewis XRESUMO
Leukocytic inflammation can be limited by inhibiting selectin-dependent leukocyte rolling. In spite of intensive efforts to develop small molecule selectin inhibitors with defined structure-activity profiles, inhibition of P-selectin-dependent leukocyte rolling in vivo by such a compound has yet to be described. We recently reported that glycosulfopeptides (GSP), modeled on the high affinity selectin ligand PSGL-1, inhibit leukocyte binding to P-selectin in vitro. Here, we have used intravital microscopy to investigate whether GSP can inhibit P-selectin-dependent leukocyte rolling in vivo. Surgical preparation of the mouse cremaster muscle for intravital microscopy induced P-selectin-dependent leukocyte rolling. Baseline rolling was recorded for 1 min followed by i.v. injection of GSP. 2-GSP-6 and 4-GSP-6 substantially reversed P-selectin-dependent leukocyte rolling, whereas control GSP, which are not fully glycosylated, did not. Inhibition of leukocyte rolling by 2- and 4-GSP-6 lasted 2-4 min. Clearance studies with 125I-labeled 4-GSP-6 demonstrated rapid reduction in its circulating levels concurrent with accumulation in urine. These data represent the first demonstration that a precisely defined structure based on a natural P-selectin ligand can inhibit P-selectin-dependent leukocyte rolling in vivo.
Assuntos
Movimento Celular/efeitos dos fármacos , Leucócitos/efeitos dos fármacos , Glicoproteínas de Membrana/química , Selectina-P/fisiologia , Peptídeos/farmacologia , Animais , Técnicas de Cultura , Relação Dose-Resposta a Droga , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiologia , Glicoproteínas/farmacologia , Cinética , Leucócitos/imunologia , Camundongos , Modelos Biológicos , EnxofreRESUMO
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.
Assuntos
Anti-Inflamatórios não Esteroides/uso terapêutico , Atorvastatina/uso terapêutico , Endotoxemia/complicações , Microvasos/efeitos dos fármacos , Óxido Nítrico Sintase Tipo II/metabolismo , Vasculite/prevenção & controle , Animais , Atorvastatina/administração & dosagem , Permeabilidade Capilar/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/enzimologia , Endotélio Vascular/fisiopatologia , Endotoxemia/enzimologia , Endotoxemia/fisiopatologia , Expressão Gênica/efeitos dos fármacos , Microscopia Intravital , Leucócitos/enzimologia , Leucócitos/fisiologia , Lipopolissacarídeos/toxicidade , Masculino , Microcirculação/efeitos dos fármacos , Microvasos/enzimologia , Óxido Nítrico Sintase Tipo II/genética , Ratos Wistar , Vasculite/induzido quimicamente , Vasculite/enzimologia , Vasculite/fisiopatologiaRESUMO
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.
Assuntos
Pulmão/microbiologia , Macrófagos Alveolares/imunologia , Óxido Nítrico/imunologia , Infecções Pneumocócicas/imunologia , Espécies Reativas de Oxigênio/imunologia , Streptococcus pneumoniae/imunologia , Animais , Apoptose/imunologia , Feminino , Imunidade Inata/imunologia , Macrófagos Alveolares/metabolismo , Glicoproteínas de Membrana/deficiência , Camundongos , Camundongos Endogâmicos C57BL , NADPH Oxidase 2 , NADPH Oxidases/deficiência , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/deficiência , Infecções Pneumocócicas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Streptococcus pneumoniae/metabolismoRESUMO
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.
Assuntos
Apoptose , Pulmão/imunologia , Macrófagos Alveolares/fisiologia , Óxido Nítrico Sintase Tipo II/deficiência , Pneumonia Pneumocócica/imunologia , Animais , Bacteriemia/enzimologia , Bacteriemia/genética , Bacteriemia/imunologia , Biomarcadores/análise , Modelos Animais de Doenças , Pulmão/enzimologia , Pulmão/microbiologia , Macrófagos Alveolares/enzimologia , Camundongos , Camundongos Mutantes , Neutrófilos/enzimologia , Neutrófilos/imunologia , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/genética , Pneumonia Pneumocócica/enzimologia , Pneumonia Pneumocócica/genética , Streptococcus pneumoniae/imunologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
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.
Assuntos
Selectina L/metabolismo , Migração e Rolagem de Leucócitos , Leucócitos/citologia , Glicoproteínas de Membrana/metabolismo , Selectina-P/metabolismo , Animais , Biotinilação , Adesão Celular , Técnicas de Cultura , Glicoproteínas/metabolismo , Leucócitos/metabolismo , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia , Microesferas , Modelos Biológicos , Oligossacarídeos/metabolismo , Ligação Proteica , Antígeno Sialil Lewis X , Fatores de TempoRESUMO
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.
Assuntos
Neutrófilos/imunologia , Neutrófilos/patologia , Pneumonia Bacteriana/imunologia , Pneumonia Bacteriana/patologia , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/patologia , Pseudomonas aeruginosa/patogenicidade , Piocianina/biossíntese , Animais , Apoptose , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Quimiocinas/biossíntese , Citocinas/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Fenazinas/metabolismo , Pneumonia Bacteriana/microbiologia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/imunologia , Pseudomonas aeruginosa/metabolismo , Piocianina/genéticaRESUMO
Cooperative action of inflammatory mediators and adhesion molecules orchestrates eosinophil recruitment during allergic inflammation in the airways. This study investigated the mechanisms involved in increasing eosinophil adhesion to human bronchial epithelial cells (HBEC) following priming and activation of eosinophils with TNF-alpha and complement protein C5a, respectively. Under primed conditions, eosinophil adhesion increased 3-fold from basal (16%), and the effect was significantly greater (p < 0.05) than the increase following stimulation with C5a alone (2-fold). Eosinophil contact with HBEC was essential for priming. In contrast to C5a, adhesion of eotaxin-stimulated eosinophils to HBEC was not primed with TNF-alpha nor IL-5, a known eosinophil-priming agent. Priming caused activation of alpha(M)beta(2) integrin; mAb against either the common beta(2) integrin subunit or its ICAM-1 ligand reduced the primed component of adhesion. Using mAbs against beta(1) or alpha(5), but not alpha(4) integrin subunit, together with anti-beta(2) integrin mAb, reduced stimulated adhesion to basal levels. Cross-linking alpha(5)beta(1) integrin increased alpha(M)beta(2) integrin-dependent adhesion of eosinophils. There are no known adhesion molecule ligands of alpha(5)beta(1) integrin expressed on HBEC; however, fibronectin, the major matrix protein ligand for alpha(5)beta(1) integrin, was detected in association with HBEC monolayers. A mAb against fibronectin, in combination with anti-beta(2) integrin mAb, reduced adhesion to basal levels. In conclusion, alpha(5)beta(1) integrin may provide a contact-dependent costimulus for eosinophil priming that, together with TNF-alpha, potentiated C5a activation of alpha(M)beta(2) integrin and increased eosinophil adhesion to ICAM-1. Fibronectin, associated with HBEC, may act as a ligand for alpha(5)beta(1) integrin. Dual regulation of eosinophil priming may prevent inappropriate activation of eosinophils in the circulation.
Assuntos
Adjuvantes Imunológicos/fisiologia , Brônquios/imunologia , Complemento C5a/fisiologia , Eosinófilos/imunologia , Receptores de Fibronectina/fisiologia , Mucosa Respiratória/imunologia , Fator de Necrose Tumoral alfa/fisiologia , Anticorpos Monoclonais/metabolismo , Antígenos CD/análise , Antígenos CD/biossíntese , Brônquios/citologia , Brônquios/metabolismo , Antígenos CD18/fisiologia , Adesão Celular/imunologia , Comunicação Celular/imunologia , Células Cultivadas , Quimiocina CCL11 , Quimiocinas CC/farmacologia , Reagentes de Ligações Cruzadas/metabolismo , Eosinófilos/metabolismo , Humanos , Integrina alfa5 , Molécula 1 de Adesão Intercelular/fisiologia , Receptores de Fibronectina/imunologia , Receptores de Fibronectina/metabolismo , Mucosa Respiratória/citologia , Mucosa Respiratória/metabolismoRESUMO
Caspase-1, the prototypic caspase, is known to process the cytokines IL-1beta and IL-18 to mature forms but it is unclear whether, like other caspases, it can induce apoptosis by activation of downstream protease cascades. Neutrophils are known to express caspase-1, to release IL-1beta and to undergo rapid, caspase-dependent apoptosis. We examined apoptosis and IL-1beta production in peripheral blood neutrophils of caspase-1-deficient and wild-type mice. Constitutive apoptosis of caspase-1-deficient neutrophils was delayed compared with wild-type neutrophils and LPS-mediated inhibition of apoptosis was absent, but caspase-1-deficient neutrophils were susceptible to Fas-mediated apoptosis. LPS-stimulated IL-1beta production was absent from caspase-1-deficient neutrophils. To ascertain whether these differences in apoptosis and IL-1beta production would alter the response to acute lung injury, we studied pulmonary neutrophil accumulation following intratracheal administration of LPS. Caspase-1-deficient mice showed increased, predominantly neutrophilic pulmonary inflammation, but inflammation had resolved in both wild-type and deficient animals by 72 h after LPS instillation. IL-1beta production was increased in wild-type lungs but was also detected in caspase-1-deficient mice. We conclude that caspase-1 modulates apoptosis of both peripheral blood and inflammatory neutrophils, but is not essential for IL-1beta production in the lung.
Assuntos
Caspase 1/deficiência , Lesão Pulmonar , Neutrófilos/enzimologia , Neutrófilos/patologia , Animais , Apoptose/efeitos dos fármacos , Caspase 1/genética , Caspase 1/imunologia , Feminino , Inflamação/enzimologia , Inflamação/imunologia , Inflamação/patologia , Interleucina-1/biossíntese , Interleucina-1/genética , Lipopolissacarídeos/toxicidade , Pulmão/enzimologia , Pulmão/imunologia , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor fas/metabolismoRESUMO
Selectin-dependent leukocyte rolling is one of the earliest steps of an acute inflammatory response and, as such, contributes to many inflammatory diseases. Although inhibiting leukocyte rolling with selectin antagonists is a strategy that promises far-reaching clinical benefit, the perceived value of this strategy has been limited by studies using inactive, weak, or poorly characterized antagonists. Recombinant P-selectin glycoprotein ligand-1-immunoglobulin (rPSGL-Ig) is a recombinant form of the best-characterized selectin ligand (PSGL-1) fused to IgG, and is one of the best prospects in the search for effective selectin antagonists. We have used intravital microscopy to investigate the ability of rPSGL-Ig to influence leukocyte rolling in living blood vessels and find that it can reduce rolling dependent on each of the selectins in vivo. Interestingly, doses of rPSGL-Ig required to reverse pre-existing leukocyte rolling are 30-fold higher than those required to limit inflammation, suggesting additional properties of this molecule. In support of this, we find that rPSGL-Ig can bind the murine chemokine KC and inhibit neutrophil migration toward this chemoattractant in vitro.