RESUMO
Neutrophils primarily act as first responders in acute infection and directly maintain inflammatory responses. However, a growing body of evidence suggests that neutrophils also bear the potential to mediate chronic inflammation by exhibiting memory-like features. We now asked whether bone marrow-derived murine neutrophils can be primed by lipoteichoic acid (LTA) from gram-positive S. aureus. We found that low-dose (1â¯ng/mL) LTA-priming promoted increased production of pro-inflammatory mediators (TNF-α, IL-6, ROS), whereas high-dose (10⯵g/mL) priming resulted in opposing reactions marked by increased IL-10 and suppressed pro-inflammatory mediators upon a second stimulus. A similar pattern of pro-inflammatory activation (trained sensitivity) and anti-inflammatory properties (tolerance) was recapitulated in cellular functional in vitro assays (transmigration and phagocytosis). Priming by LTA correlated with TLR2/MyD88-mediated regulation of NFκB-p65 through intermediate PI3Ks/MAPK. Collectively, our data suggest a previously unknown capacity of neutrophils to be differentially primed by varying doses of LTA, endorsing memory-like features in neutrophils.
Assuntos
Neutrófilos , Staphylococcus aureus , Animais , Medula Óssea , Mediadores da Inflamação , Lipopolissacarídeos/farmacologia , Camundongos , Ácidos Teicoicos/farmacologiaRESUMO
A growing body of evidence suggests that innate immune cells can respond in a memory-like (adaptive) fashion, which is referred to as trained immunity. Only few in vivo studies have shown training effects in neutrophils; however, no in vitro setup has been established to study the induction of trained immunity or tolerance in neutrophils by microbial agents. In light of their short lifespan (up to 48 h), we suggest to use the term trained sensitivity for neutrophils in an in vitro setting. Here, we firstly describe a feasible two-hit model, using different doses of lipopolysaccharide (LPS) in bone marrow neutrophils. We found that low doses (10 pg/mL) induce pro-inflammatory activation (trained sensitivity), whereas priming with high doses (100 ng/mL) leads to suppression of pro-inflammatory mediators such as TNF-α or IL-6 (tolerance) (p < 0.05). On a functional level, trained neutrophils displayed increased phagocytic activity and LFA-1 expression as well as migrational capacity and CD11a expression, whereas tolerant neutrophils show contrasting effects in vitro. Mechanistically, TLR4/MyD88/PI3Ks regulate the activation of p65, which controls memory-like responses in mouse bone marrow neutrophils (p < 0.05). Our results open a new window for further in vitro studies on memory-like inflammatory responses of short-lived innate immune cells such as neutrophils.
Assuntos
Imunidade Inata/efeitos dos fármacos , Memória Imunológica/efeitos dos fármacos , Mediadores da Inflamação/imunologia , Neutrófilos/efeitos dos fármacos , Animais , Medula Óssea/efeitos dos fármacos , Medula Óssea/imunologia , Células Cultivadas , Citocinas/genética , Citocinas/imunologia , Tolerância Imunológica/efeitos dos fármacos , Tolerância Imunológica/imunologia , Memória Imunológica/imunologia , Lipopolissacarídeos/toxicidade , Camundongos , Neutrófilos/imunologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologiaRESUMO
BACKGROUND: A growing body of evidence defines inflammation as a hallmark feature of disease pathogenesis of Duchenne muscular dystrophy. To tailor potential immune modulatory interventions, a better understanding of immune dysregulation in Duchenne muscular dystrophy is needed. We now asked whether dystrophin deficiency affects the cascade of leukocyte recruitment. METHODS: We performed intravital microscopy on the cremaster muscle of wild-type and dystrophin-deficient mdx mice. Recruitment was triggered by preparation alone (traumatic inflammation) or in combination with scrotal TNFα injections. Neutrophilic infiltration of the cremaster muscle was assessed on tissue sections. Integrin expression on circulating neutrophils and serum levels of pro-inflammatory cytokines were measured by flow cytometry. RESULTS: Mdx mice show increased rolling and adhesion at baseline (traumatic inflammation) and a more profound response upon TNFα injection compared with wild-type animals. In both models, neutrophilic infiltration of the cremaster muscle is increased. Upregulation of the integrins LFA-1 and Mac-1 on circulating leukocytes and pro-inflammatory cytokines IL-6 and CCL2 in the serum points toward systemically altered immune regulation in mdx mice. CONCLUSION: We are the first to show exaggerated activation of the leukocyte recruitment cascade in a dystrophin-deficient organism in vivo.
Assuntos
Distrofina/deficiência , Migração e Rolagem de Leucócitos , Leucócitos/citologia , Distrofia Muscular de Duchenne/imunologia , Músculos Abdominais/metabolismo , Animais , Citocinas/metabolismo , Modelos Animais de Doenças , Distrofina/metabolismo , Citometria de Fluxo , Inflamação , Integrinas/metabolismo , Microscopia Intravital , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Músculo Esquelético/patologia , Neutrófilos/metabolismo , Escroto/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Although fish oil-based and olive oil-based lipid emulsions have been shown to exert anti-inflammatory functions, the immunomodulating properties of lipids are still controversial. Therefore, we investigated the anti-inflammatory effect of three different parenterally administered lipid emulsions in vivo: olive oil-based Clinoleic, fish oil-based Smoflipid, and soybean oil-based Lipofundin. We observed leukocyte recruitment in inflamed murine cremaster muscle using intravital microscopy and survival in a murine model of LPS-induced systemic inflammation and analyzed expression of leukocyte and endothelial adhesion molecules. Olive oil-based Clinoleic and fish oil-based Smoflipid profoundly inhibited leukocyte adhesion compared to Lipofundin during LPS-induced inflammation of the murine cremaster muscle. In the trauma model of cremaster muscle inflammation, Lipofundin was the only lipid emulsion that even augmented leukocyte adhesion. In contrast to Smoflipid and Lipofundin, Clinoleic effectively blocked leukocyte recruitment and increased survival during lethal endotoxemia. Flow chamber experiments and analysis of adhesion molecule expression suggest that both endothelial and leukocyte driven mechanisms might contribute to anti-inflammatory effects of Clinoleic. We conclude that the anti-inflammatory properties of Clinoleic are superior to those of Smoflipid and Lipofundin even during systemic inflammation. Thus, these results should stimulate further studies investigating parenteral lipids as an anti-inflammatory strategy in critically ill patients.
Assuntos
Leucócitos/citologia , Leucócitos/efeitos dos fármacos , Azeite de Oliva/química , Óleos de Plantas/química , Óleos de Plantas/farmacologia , Óleo de Soja/química , Óleo de Soja/farmacologia , Animais , Células Cultivadas , Óleos de Peixe/química , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Músculos/citologia , Músculos/efeitos dos fármacosRESUMO
As nuclear factor kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) seem to be critical mediators in the inflammatory response, we studied the effects of lipopolysaccharide (LPS) and lipoteichoic acid (LTA) on (a) the activation of NF-κB and MAPKs and (b) the expression of tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) with or without the specific inhibitors of these intracellular signal transduction pathways in neonatal cord and adult blood. TNF-α and IL-6 concentrations showed a sharp increase in the supernatants of cord and adult whole blood after stimulation. TNF-α concentrations were significantly higher, whereas IL-6 concentrations were tendentially lower in adult blood after stimulation. Stimulation with LPS or LTA resulted in a significantly decreased activation of p38 MAPK in neonatal compared with adult blood. Although LTA failed to induce additional ERK1/2 phosphorylation, LPS stimulation mediated the moderately increased levels of activated ERK1/2 in neonatal monocytes. The addition of the p38 MAPK inhibitor SB202190 significantly decreased IL-6 and TNF-α production upon LPS or LTA stimulation. Furthermore, the inhibition of ERK1/2 was able to reduce LPS-stimulated TNF-α production in neonatal blood. We conclude that p38 MAPK as well as ERK1/2 phosphorylation is crucially involved in LPS activation and could explain the differences in early cytokine response between neonatal and adult blood.
Assuntos
Regulação da Expressão Gênica , Interleucina-6/sangue , Lipopolissacarídeos/química , NF-kappa B/sangue , Ácidos Teicoicos/química , Fator de Necrose Tumoral alfa/sangue , Adulto , Feminino , Sangue Fetal/metabolismo , Citometria de Fluxo , Humanos , Imidazóis/química , Recém-Nascido , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Sistema de Sinalização das MAP Quinases , Monócitos/citologia , Fosforilação , Gravidez , Piridinas/química , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
By binding ß 2-integrins both ICAM-1 and the receptor for advanced glycation end products (RAGE) mediate leukocyte recruitment in a stimulus-dependent manner. Using different inflammatory mouse models we investigated how RAGE and ICAM-1 are involved in anti-inflammatory functions of protein C (PC; Ceprotin, 100 U/kg). We found that, depending on the stimulus, RAGE and ICAM-1 are cooperatively involved in PC-induced inhibition of leukocyte recruitment in cremaster models of inflammation. During short-term proinflammatory stimulation (trauma, fMLP, and CXCL1), ICAM-1 is more important for mediation of anti-inflammatory effects of PC, whereas RAGE plays a major role after longer proinflammatory stimulation (TNF α ). In contrast to WT and Icam-1(-/-) mice, PC had no effect on bronchoalveolar neutrophil emigration in RAGE(-/-) mice during LPS-induced acute lung injury, suggesting that RAGE critically mediates PC effects during acute lung inflammation. In parallel, PC treatment effectively blocked leukocyte recruitment and improved survival of WT mice and Icam-1-deficient mice in LPS-induced endotoxemia, but failed to do so in RAGE-deficient mice. Exploring underlying mechanisms, we found that PC is capable of downregulating intracellular RAGE and extracellular ICAM-1 in endothelial cells. Taken together, our data show that RAGE and ICAM-1 are required for the anti-inflammatory functions of PC.
Assuntos
Inflamação/metabolismo , Molécula 1 de Adesão Intercelular/fisiologia , Proteína C/metabolismo , Receptores Imunológicos/fisiologia , Lesão Pulmonar Aguda/patologia , Animais , Coagulação Sanguínea , Modelos Animais de Doenças , Células Endoteliais/citologia , Endotoxemia/patologia , Leucócitos/citologia , Lipopolissacarídeos/química , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Músculo Esquelético/patologia , Receptor para Produtos Finais de Glicação Avançada , Fator de Necrose Tumoral alfa/metabolismoRESUMO
AIMS: The receptor for advanced glycation endproducts, RAGE, is a multiligand receptor and NF-κB activator leading to perpetuation of inflammation. We investigated whether and how RAGE is involved in mediation of anti-inflammatory properties of protein C. METHODS AND RESULTS: We analyzed the effect of protein C on leukocyte adhesion and transmigration in WT- and RAGE-deficient mice using intravital microscopy of cremaster muscle venules during trauma- and TNFα-induced inflammation. Both, protein C (PC, Ceprotin, 100 U/kg) and activated protein C (aPC, 24 µg/kg/h) treatment significantly inhibited leukocyte adhesion in WT mice in these inflammation models. The impaired leukocyte adhesion after trauma-induced inflammation in RAGE knockout mice could not be further reduced by PC and aPC. After TNFα-stimulation, however, aPC but not PC treatment effectively blocked leukocyte adhesion in these mice. Consequently, we asked whether RAGE is involved in PC activation. Since RAGE-deficient mice and endothelial cells showed insufficient PC activation, and since thrombomodulin (TM) and endothelial protein C receptor (EPCR) are reduced on the mRNA and protein level in RAGE deficient endothelial cells, an involvement of RAGE in TM-EPCR-dependent PC activation is likely. Moreover, TNFα-induced activation of MAPK and upregulation of ICAM-1 and VCAM-1 are reduced both in response to aPC treatment and in the absence of RAGE. Thus, there seems to be interplay of the RAGE and the PC pathway in inflammation. CONCLUSION: RAGE controls anti-inflammatory properties and activation of PC, which might involve EPCR and TM.
Assuntos
Anti-Inflamatórios/metabolismo , Proteína C/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores Imunológicos/metabolismo , Transdução de Sinais/genética , Animais , Adesão Celular/genética , Células Endoteliais/metabolismo , Receptor de Proteína C Endotelial , Humanos , Inflamação/genética , Inflamação/metabolismo , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Leucócitos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína C/genética , Receptor para Produtos Finais de Glicação Avançada , Receptores de Superfície Celular/genética , Receptores Imunológicos/genética , Trombomodulina/genética , Trombomodulina/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Regulação para Cima/genética , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
BACKGROUND: Human milk contains immune molecules involved in the protection of newborns against infections. We analyzed the concentration of Deleted in Malignant Brain Tumors 1 (DMBT1), a protein with functions in innate immunity, in breast milk. METHODS: DMBT1 was detected in breast milk by Western blotting and its concentration was quantified by ELISA in 95 breast milk samples collected from mothers of preterm and term neonates during the first four weeks after delivery. Possible effects of maternal or neonatal parameters were analyzed by different statistical tests. RESULTS: The mean DMBT1 concentration (± standard error of the mean) in the tested milk samples was 2.48 ± 0.26 µg/mL (range: 0.112 µg/mL to 17.984 µg/mL) and represented 0.0087% of the total protein content. The comparison between the newborns with infection and the newborns without infection revealed significantly higher DMBT1 concentrations in breast milk in the group with infection (6.72 ± 2.53 µg/mL versus 2.20 ± 0.35 µg/mL (P = 0.031)). Neither maternal nor neonatal parameters showed a correlation with the milk DMBT1 levels. CONCLUSIONS: DMBT1 is a component of breast milk after birth and is up-regulated in the breast milk from mothers with newborns suffering from neonatal infection. Thus, breast milk DMBT1 may be part of the innate immunity similar to secretory IgA.
Assuntos
Leite Humano/imunologia , Receptores de Superfície Celular/metabolismo , Infecções Estafilocócicas/imunologia , Infecções Estreptocócicas/imunologia , Adulto , Biomarcadores/metabolismo , Western Blotting , Proteínas de Ligação ao Cálcio , Proteínas de Ligação a DNA , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imunidade Inata , Recém-Nascido , Recém-Nascido Prematuro , Doenças do Prematuro/imunologia , Modelos Lineares , Modelos Logísticos , Masculino , Leite Humano/metabolismo , Estudos Prospectivos , Proteínas Supressoras de Tumor , Regulação para CimaRESUMO
It is well acknowledged that proinflammatory stimulation during acute hyperglycemia is able to aggravate inflammatory diseases. However, the mechanisms of proinflammatory effects of glucose are controversially discussed. We investigated leukocyte recruitment after intravenous injection of glucose in different inflammatory models using intravital microscopy. Flow chamber experiments, expression analysis, functional depletion, and knockout of key adhesion molecules gave mechanistic insight in involved pathways. We demonstrated that a single injection of glucose rapidly increased blood glucose levels in a dose-dependent manner. Notably, during tumor necrosis factor (TNF) α-induced inflammation leukocyte recruitment was not further enhanced by glucose administration, whereas glucose injection profoundly augmented leukocyte adhesion and transmigration into inflamed tissue in the trauma model, indicating that proinflammatory properties of glucose are stimulus dependent. Experiments with functional or genetic inhibition of the chemokine receptor CXCR2, intercellular adhesion molecule 1 (ICAM1), and lymphocyte function antigen 1 (LFA1) suggest that keratino-derived-chemokine CXCL1-triggered interactions of ICAM1 and LFA1 are crucially involved in the trauma model of inflammation. The lacking effect of glucose on ß(2) integrin expression and on leukocyte adhesion in dynamic flow chamber experiments argues against leukocyte-driven underlying mechanisms and favours an endothelial pathway since endothelial ICAM1 expression was significantly upregulated in response to glucose.
Assuntos
Quimiocina CXCL1/farmacologia , Glucose/farmacologia , Inflamação/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Antígeno-1 Associado à Função Linfocitária/metabolismo , Animais , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Citometria de Fluxo , Imuno-Histoquímica , Molécula 1 de Adesão Intercelular/genética , Leucócitos/citologia , Antígeno-1 Associado à Função Linfocitária/genética , Camundongos , Camundongos Knockout , Ligação Proteica/efeitos dos fármacosRESUMO
BACKGROUND: The receptor for advanced glycation endproducts, RAGE, is involved in the pathogenesis of many inflammatory conditions, which is mostly related to its strong activation of NF-κB but also due to its function as ligand for the ß2-integrin Mac-1. To further dissect the stimulus-dependent role of RAGE on leukocyte recruitment during inflammation, we investigated ß2-integrin-dependent leukocyte adhesion in RAGE-/- and Icam1-/- mice in different cremaster muscle models of inflammation using intravital microscopy. RESULTS: We demonstrate that RAGE, but not ICAM-1 substantially contributes to N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced leukocyte adhesion in TNF-α-pretreated cremaster muscle venules in a Mac-1-dependent manner. In contrast, fMLP-stimulated leukocyte adhesion in unstimulated cremaster muscle venules is independent of RAGE, but dependent on ICAM-1 and its interaction with LFA-1. Furthermore, chemokine CXCL1-stimulated leukocyte adhesion in surgically prepared cremaster muscle venules was independent of RAGE but strongly dependent on ICAM-1 and LFA-1 suggesting a differential and stimulus-dependent regulation of leukocyte adhesion during inflammation in vivo. CONCLUSION: Our results demonstrate that RAGE and ICAM-1 differentially regulate leukocyte adhesion in vivo in a stimulus-dependent manner.
Assuntos
Molécula 1 de Adesão Intercelular/metabolismo , Leucócitos/metabolismo , Antígeno-1 Associado à Função Linfocitária/metabolismo , Receptores Imunológicos/metabolismo , Vênulas/metabolismo , Animais , Adesão Celular/genética , Adesão Celular/imunologia , Células Cultivadas , Quimiocina CXCL1/metabolismo , Inflamação , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/imunologia , Leucócitos/imunologia , Leucócitos/patologia , Antígeno-1 Associado à Função Linfocitária/imunologia , Antígeno de Macrófago 1/imunologia , Antígeno de Macrófago 1/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculos/patologia , N-Formilmetionina Leucil-Fenilalanina/metabolismo , Ligação Proteica/genética , Ligação Proteica/imunologia , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/genética , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Vênulas/patologiaRESUMO
Anti-inflammatory properties of protein C (PC) concentrate are poorly studied compared to activated protein C, although PC is suggested to be safer in clinical use. We investigated how PC interferes with the leukocyte recruitment cascade during acute inflammation and its efficacy during murine endotoxemia. We found that similar to activated protein infusion, intravenous PC application reduced leukocyte recruitment in inflamed tissues in a dose- and time-dependent manner. During both tumor necrosis factor-α induced and trauma-induced inflammation of the cremaster muscle, intravital microscopy revealed that leukocyte adhesion and transmigration, but not rolling, were profoundly inhibited by 100 U/kg PC. Moreover, PC blocked leukocyte emigration into the bronchoalveolar space during lipopolysaccharide (LPS) induced acute lung injury. PC was efficiently activated in a murine endotoxemia model, which reduced leukocyte infiltration of organs and strongly improved survival (75% versus 25% of control mice). Dependent on the inflammatory model, PC provoked a significant inhibition of leukocyte recruitment as early as 1 hour after administration. PC-induced inhibition of leukocyte recruitment during acute inflammation critically involves thrombomodulin-mediated PC activation, subsequent endothelial PC receptor and protease-activated receptor-1-dependent signaling, and down-regulation of intercellular adhesion molecule 1 leading to reduced endothelial inflammatory response. We conclude that during acute inflammation and sepsis, PC is a fast acting and effective therapeutic approach to block leukocyte recruitment and improve survival.
Assuntos
Lesão Pulmonar Aguda/imunologia , Anti-Inflamatórios/farmacologia , Endotoxemia/imunologia , Leucócitos/efeitos dos fármacos , Pneumonia/imunologia , Proteína C/farmacologia , Animais , Antibacterianos/farmacologia , Antígenos CD/metabolismo , Adesão Celular/imunologia , Movimento Celular/imunologia , Quimiotaxia de Leucócito/efeitos dos fármacos , Quimiotaxia de Leucócito/imunologia , Citocinas/metabolismo , Relação Dose-Resposta a Droga , Receptor de Proteína C Endotelial , Endotoxemia/tratamento farmacológico , Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/imunologia , Molécula 1 de Adesão Intercelular/fisiologia , Lipopolissacarídeos/toxicidade , Antígeno-1 Associado à Função Linfocitária/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Músculo Liso Vascular/imunologia , Músculo Liso Vascular/lesões , Miosite/imunologia , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Análise de Sobrevida , Trombomodulina/metabolismo , Fator de Necrose Tumoral alfa/toxicidadeRESUMO
BACKGROUND: Sepsis continues to be a leading cause of morbidity and mortality in newborns. OBJECTIVE: As both nuclear factor-kappa B (NF-κB) and p38 mitogen-activated protein kinase (MAPK) appear to be critical mediators in inflammatory response, we studied the effects of lipopolysaccharide (LPS) on expression and function of NF-κB and p38 MAPK in whole neonatal cord and adult blood. METHODS: Th1/Th2 cytokine concentrations and phosphorylation of NF-κB and p38 MAPK were determined by flow-cytometric analysis. RESULTS: Tumor necrosis factor-alpha (TNF-α), IL-6, and IL-10 concentrations were significantly elevated in supernatants of neonatal and adult blood after LPS stimulation for 4 h. IFN-γ, IL-4, and IL-2 showed no significant alterations. Furthermore, TNF-α concentrations were significantly higher in adult compared to neonatal blood after LPS stimulation. Stimulation with LPS resulted in significantly decreased activation of p38 MAPK in neonatal blood, whereas NF-κB showed no difference. Following inhibition of p38 MAPK with the specific inhibitor SB-202190, levels of TNF-α and IL-6 significantly decreased in neonatal and adult blood, whereas pharmacological inhibition of NF-κB with SC-514 showed no significant effect on cytokine expression. CONCLUSIONS: We conclude that p38 MAPK phosphorylation is crucially involved in LPS activation and could explain the differences in early cytokine response between neonatal and adult blood.
Assuntos
Citocinas/biossíntese , Sangue Fetal/imunologia , Lipopolissacarídeos/farmacologia , Células Th1/metabolismo , Células Th2/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/biossíntese , Adulto , Citocinas/sangue , Inibidores Enzimáticos/farmacologia , Sangue Fetal/efeitos dos fármacos , Sangue Fetal/enzimologia , Citometria de Fluxo , Humanos , Imidazóis/farmacologia , Recém-Nascido , NF-kappa B/antagonistas & inibidores , NF-kappa B/biossíntese , NF-kappa B/sangue , Fosforilação/efeitos dos fármacos , Piridinas/farmacologia , Transdução de Sinais , Células Th1/efeitos dos fármacos , Células Th1/enzimologia , Células Th1/imunologia , Células Th2/efeitos dos fármacos , Células Th2/enzimologia , Células Th2/imunologia , Tiofenos/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/sangueRESUMO
The receptor for advanced glycation end products (RAGE) contributes to the inflammatory response in many acute and chronic diseases. In this context, RAGE has been identified as a ligand for the beta(2)-integrin Mac-1 under static in vitro conditions. Because intercellular adhesion molecule (ICAM)-1 also binds beta(2)-integrins, we studied RAGE(-/-), Icam1(-/-), and RAGE(-/-) Icam1(-/-) mice to define the relative contribution of each ligand for leukocyte adhesion in vivo. We show that trauma-induced leukocyte adhesion in cremaster muscle venules is strongly dependent on RAGE and ICAM-1 acting together in an overlapping fashion. Additional in vivo experiments in chimeric mice lacking endothelium-expressed RAGE and ICAM-1 located the adhesion defect to the endothelial compartment. Using microflow chambers coated with P-selectin, CXCL1, and soluble RAGE (sRAGE) demonstrated that sRAGE supports leukocyte adhesion under flow conditions in a Mac-1- but not LFA-1-dependent fashion. A static adhesion assay revealed that wild-type and RAGE(-/-) neutrophil adhesion and spreading were similar on immobilized sRAGE or fibrinogen. These observations indicate a crucial role of endothelium-expressed RAGE as Mac-1 ligand and uncover RAGE and ICAM-1 as a new set of functionally linked adhesion molecules, which closely cooperate in mediating leukocyte adhesion during the acute trauma-induced inflammatory response in vivo.
Assuntos
Quimiotaxia de Leucócito/fisiologia , Endotélio Vascular/metabolismo , Molécula 1 de Adesão Intercelular/fisiologia , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Músculo Esquelético/irrigação sanguínea , Vasculite/imunologia , Doença Aguda , Animais , Adesão Celular , Forma Celular , Quimiotaxia de Leucócito/efeitos dos fármacos , Humanos , Leucotrieno B4/farmacologia , Ligantes , Antígeno de Macrófago 1/metabolismo , Camundongos , Camundongos Knockout , Proteínas Quinases Ativadas por Mitógeno/deficiência , Músculo Esquelético/lesões , Músculo Esquelético/patologia , Neutrófilos/patologia , Quimera por Radiação , Proteínas Recombinantes de Fusão/fisiologia , Fator de Necrose Tumoral alfa/farmacologia , Vasculite/etiologia , Vênulas/patologiaRESUMO
BACKGROUND: Clinical studies failed to provide clear evidence for a proatherogenic role of hypercoagulability. This is in contrast to the well-established detrimental role of hypercoagulability and thrombin during acute atherosclerotic complications. These seemingly opposing data suggest that hypercoagulability might exert both proatherogenic and antiatherogenic effects. We therefore investigated whether hypercoagulability mediates a beneficial effect during de novo atherogenesis. METHODS AND RESULTS: De novo atherogenesis was evaluated in 2 mouse models with hyperlipidemia and genetically imposed hypercoagulability (TM(Pro/Pro)ApoE(-/-) and FVL(Q/Q)ApoE(-/-) mice). In both mouse models, hypercoagulability resulted in larger plaques, but vascular stenosis was not enhanced secondary to positive vascular remodeling. Importantly, plaque stability was increased in hypercoagulable mice with less necrotic cores, more extracellular matrix, more smooth muscle cells, and fewer macrophages. Long-term anticoagulation reversed these changes. The reduced frequency of intraplaque macrophages in hypercoagulable mice is explained by an inhibitory role of thrombin and protease-activated receptor-1 on monocyte transendothelial migration in vitro. This is dependent on phospholipase-Cbeta, phosphoinositide 3-kinase, and nitric oxide signaling in monocytes but not in endothelial cells. CONCLUSIONS: Here, we show a new function of the coagulation system, averting stenosis and plaque destabilization during de novo atherogenesis. The in vivo and in vitro data establish that thrombin-induced signaling via protease-activated receptor-1, phospholipase-Cbeta, phosphoinositide 3-kinase, and nitric oxide in monocytes impairs monocyte transendothelial migration. This likely accounts for the reduced macrophage accumulation in plaques of hypercoagulable mice. Thus, in contrast to their role in unstable plaques or after vascular injury, hypercoagulability and thrombin convey a protective effect during de novo atherogenesis.
Assuntos
Aterosclerose/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfolipase C beta/metabolismo , Receptor PAR-1/metabolismo , Trombofilia/metabolismo , Animais , Apolipoproteínas E/genética , Aterosclerose/imunologia , Aterosclerose/patologia , Movimento Celular/imunologia , Endotélio Vascular/imunologia , Endotélio Vascular/metabolismo , Feminino , Hiperlipidemias/imunologia , Hiperlipidemias/metabolismo , Hiperlipidemias/patologia , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Monócitos/citologia , Monócitos/metabolismo , Óxido Nítrico/metabolismo , Transdução de Sinais/fisiologia , Trombina/metabolismo , Trombofilia/imunologia , Vasculite/imunologia , Vasculite/metabolismo , Vasculite/patologiaRESUMO
The mammalian actin-binding protein 1 (mAbp1, Hip-55, SH3P7) is phosphorylated by the nonreceptor tyrosine kinase Syk that has a fundamental effect for several beta(2) integrin (CD11/CD18)-mediated neutrophil functions. Live cell imaging showed a dynamic enrichment of enhanced green fluorescence protein-tagged mAbp1 at the phagocytic cup of neutrophil-like differentiated HL-60 cells during beta(2) integrin-mediated phagocytosis of serum-opsonized Escherichia coli. The genetic absence of Syk or its pharmacologic inhibition using piceatannol abrogated the proper localization of mAbp1 at the phagocytic cup. The genetic absence or down-regulation of mAbp1 using the RNA interference technique significantly compromised beta(2) integrin-mediated phagocytosis of serum-opsonized E coli or Salmonella typhimurium in vitro as well as clearance of S typhimurium infection in vivo. Moreover, the genetic absence of mAbp1 almost completely abrogated firm neutrophil adhesion under physiologic shear stress conditions in vitro as well as leukocyte adhesion and extravasation in inflamed cremaster muscle venules of mice treated with tumor-necrosis factor alpha. Functional analysis showed that the down-regulation of mAbp1 diminished the number of beta(2) integrin clusters in the high-affinity conformation under flow conditions. These unanticipated results define mAbp1 as a novel molecular player in integrin biology that is critical for phagocytosis and firm neutrophil adhesion under flow conditions.
Assuntos
Antígenos CD18/fisiologia , Proteínas dos Microfilamentos/fisiologia , Neutrófilos/fisiologia , Domínios de Homologia de src/fisiologia , Animais , Adesão Celular/fisiologia , Escherichia coli/patogenicidade , Células HL-60 , Humanos , Técnicas In Vitro , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Mutantes , Proteínas dos Microfilamentos/antagonistas & inibidores , Proteínas dos Microfilamentos/deficiência , Proteínas dos Microfilamentos/genética , Fagocitose/fisiologia , Proteínas Tirosina Quinases/deficiência , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Interferência de RNA , Receptores de IgG/metabolismo , Salmonella typhimurium/patogenicidade , Quinase Syk , Domínios de Homologia de src/genéticaRESUMO
beta(2) integrins and Fcgamma receptors are critically involved in neutrophil activation at the site of inflammation. Both receptor types trigger a receptor-proximal tyrosine phosphorylation cascade through Src family kinases and Syk, but further downstream signaling events are poorly understood. We show that phospholipase C (PLC) gamma2 is phosphorylated downstream of Src family kinases and Syk during integrin or Fc receptor-mediated activation of neutrophils. PLCgamma2(-/-) neutrophils are completely defective in beta(2) integrin or Fcgamma receptor-mediated functional responses such as respiratory burst, degranulation, or cell spreading in vitro and show reduced adhesion/spreading in inflamed capillary venules in vivo. However, PLCgamma2(-/-) neutrophils respond normally to various other agonists, including chemokines, bacterial formyl peptides, Toll-like receptor ligands, or proinflammatory cytokines, and migrate normally both in vitro and in vivo. To confirm the in vivo relevance of these observations, the effect of the PLCgamma2(-/-) mutation was tested in the K/BxN serum transfer arthritis model, which is known to require beta(2) integrins, Fcgamma receptors, and neutrophils. PLCgamma2 deficiency completely protected mice from clinical signs and histological features of arthritis as well as from arthritis-induced loss of articular function. These results identify PLCgamma2 as a critical player of integrin and Fc receptor-mediated neutrophil functions and the neutrophil-mediated effector phase of autoimmune arthritis.
Assuntos
Artrite/enzimologia , Doenças Autoimunes/enzimologia , Antígenos CD18/metabolismo , Neutrófilos/citologia , Neutrófilos/enzimologia , Fosfolipase C gama/metabolismo , Receptores de IgG/metabolismo , Animais , Artrite/complicações , Artrite/patologia , Artrite/prevenção & controle , Doenças Autoimunes/complicações , Doenças Autoimunes/patologia , Doenças Autoimunes/prevenção & controle , Biomarcadores/metabolismo , Medula Óssea/efeitos dos fármacos , Medula Óssea/enzimologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Movimento Celular/efeitos dos fármacos , Quimera , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Isoenzimas/metabolismo , Leucócitos/citologia , Leucócitos/efeitos dos fármacos , Camundongos , Músculos/efeitos dos fármacos , Músculos/metabolismo , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/efeitos dos fármacos , Fosfolipase C gama/deficiência , Fosforilação/efeitos dos fármacos , Proteínas Tirosina Quinases/metabolismo , Receptores de Citocinas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/efeitos dos fármacos , Quinase Syk , Quinases da Família src/metabolismoRESUMO
BACKGROUND: During inflammation, beta2-integrins mediate leukocyte adhesion to the endothelium accompanied by the activation of the spleen tyrosine kinase Syk. RESULTS: We investigated leukocyte adhesion and rolling in cremaster muscle venules before and during stimulation with fMLP using mice with a Syk-/- hematopoietic system. In unstimulated venules, Syk-/- leukocytes adhered less efficiently than control leukocytes while rolling was similar between Syk-/- and control leukocytes. During fMLP-superfusion, control mice showed significantly increased adhesion accompanied by reduced rolling. For Syk-/- leukocytes, an increase in adhesion with a concomitant decrease in rolling was only observed during the first three minutes during fMLP stimulation, but not at later time points. We also investigated leukocyte spreading against the vessel wall during fMLP stimulation and found a significant impairment of spreading for Syk-/- leukocytes. Additional in vitro experiments revealed that the adhesion and spreading defect seen in Syk-/- chimeric mice was due to compromised beta2-integrin-mediated outside-in signaling. CONCLUSION: We provide substantial evidence for an important role of Syk in mediating beta2-integrin dependent outside-in signaling leading to sustained leukocyte adhesion and spreading during the inflammatory response in vivo.
Assuntos
Adesão Celular/imunologia , Inflamação/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Migração e Rolagem de Leucócitos/fisiologia , Proteínas Tirosina Quinases/metabolismo , Animais , Antígenos CD18/imunologia , Antígenos CD18/metabolismo , Endotélio Vascular/imunologia , Endotélio Vascular/metabolismo , Processamento de Imagem Assistida por Computador , Inflamação/induzido quimicamente , Inflamação/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Camundongos , Camundongos Mutantes , Músculo Esquelético/irrigação sanguínea , N-Formilmetionina Leucil-Fenilalanina/toxicidade , Proteínas Tirosina Quinases/imunologia , Quinase Syk , Quimeras de Transplante , Vênulas/imunologiaRESUMO
Leukocyte adhesion via beta(2) integrins (CD11/CD18) activates the tyrosine kinase Syk. We found that Syk was enriched at the lamellipodium during N-formyl-Met-Leu-Phe-induced migration of neutrophil-like differentiated HL-60 cells. Here, Syk colocalized with Vav, a guanine nucleotide exchange factor for Rac and Cdc42. The enrichment of Syk at the lamellipodium and its colocalization with Vav were absent upon expression of a Syk kinase-dead mutant (Syk K402R) or a Syk mutant lacking the binding site of Vav (Syk Y348F). Live cell imaging revealed that both mutations resulted in excessive lamellipodium formation and severely compromised migration compared with control cells. Similar results were obtained upon down-regulation of Syk by RNA interference (RNAi) technique as well as in Syk(-/-) neutrophils from wild-type mice reconstituted with Syk(-/-) bone marrow. A pivotal role of Syk in vivo was demonstrated in the Arthus reaction, where neutrophil extravasation, edema formation, and hemorrhage were profoundly diminished in Syk(-/-) bone marrow chimeras compared with those in control animals. In the inflamed cremaster muscle, Syk(-/-) neutrophils revealed a defect in adhesion and migration. These findings indicate that Syk is critical for beta(2) integrin-mediated neutrophil migration in vitro and plays a fundamental role in neutrophil recruitment during the inflammatory response in vivo.